Compositions and methods for treatment of autoimmune and inflammatory diseases and disorders

ABSTRACT

The present application discloses a novel fusion peptide of IL-2 and IL-33 and its use. It comprises a biologically active domain of Interleukin-2 (IL-2) or a biologically active fragment or homolog thereof, and a biologically active domain of Interleukin-33 (IL-33) or a biologically active fragment or homolog thereof. The two portions can be linked by a linker sequence. The application discloses that combination therapies using IL-2 and IL-33 or a therapy using the IL233 fusion protein are effective in preventing or treating diseases and disorders such as autoimmune diseases and disorders, inflammation, etc. Depending on the subject&#39;s disease or disorder, the compositions of the invention are useful for preventing certain symptoms, treating the disease, and alleviating at least some of the symptoms.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.15/680,320, filed Aug. 18, 2017 (now U.S. Pat. No. 10,851,145), whichitself is continuation of U.S. patent application Ser. No. 15/026,927,filed Apr. 1, 2016 (now U.S. Pat. No. 9,840,545), which itself is a U.S.National Stage Entry of PCT International Patent Application Serial No.PCT/US2014/056767, filed Sep. 22, 2014, which itself claimed prioritypursuant to 35 U.S.C. § 119(e) to U.S. Provisional Application Ser. No.61/880,257, filed Sep. 20, 2013. The entire disclosure of each of theaforementioned patent applications is incorporated herein by reference.

BACKGROUND

Although, IL-2 was discovered more than three decades ago, novelfunctions of this pleiotropic cytokine are still being discovered (1).These include the role of IL-2 in maintenance of immune tolerance andregulation of T-helper cell (Th) function for organ-specificity ofinflammation (14-18). The current invention pertains to the utilizationof IL-2 in conjunction with a recently described cytokine IL-33.

Regulatory T-cells and IL-2: The Foxp3⁺CD4⁺ Regulatory T-cells (Treg)are important for peripheral tolerance and the deficiency of Treg cellshas been demonstrated to be an underlying factor for several autoimmuneand inflammatory diseases (3-5). The differentiation, survival and thefunction of the Treg cells is critically dependent on Interleukin-2(IL-2) (19-22). Mice that are deficient in IL-2 have reduced proportionsand numbers of Treg cells. Consequently, there is uncontrolledactivation of immune cells leading to a lymphoproliferative disorder,multi-organ inflammation and death between 1 to 4 months of lifecompared to the normal mice (14-16, 18).

IL2 and Th2 cells: IL-2 is produced by the immune cells (mainly T-cells)and supports the homeostasis and function of another important cell-typeof the immune system known as Th2 cells. IL-2 regulates the developmentand function of the Th2 cells in vivo (14, 16). The primary function ofthe Th2 cells is to fight parasitic infections and boost the productionof anti-parasitic antibodies by the B-cells. However, they have alsobeen known to suppress organ-specific inflammation during autoimmunity,observed in type 1 diabetes (T1D; also referred to as diabetes mellitustype 1) and multiple sclerosis (MS) (23-26). The Th2 cells have alsobeen recognized as potent suppressors of Th1 cells and cytolytic T-cellsthat induce organ rejection during transplantation (27-31).

Treg independent anti-inflammatory functions of IL-2: IL-2 has beenshown to be important negative regulator of inflammation via regulatingTreg homeostasis and function. Recent studies demonstrated the importantfunction of IL-2 in preventing organ-specific inflammation independentof its role in Treg cells, via regulating the phenotype and function ofthe Th cells, which in-turn regulates the B-cell responses. The IL2′(IL2 knock-out (1(0)) mice have much reduced Tregs that leads tospontaneous inflammation in pancreas, salivary glands, and liver(14-16). However, the Scurfy (Sf) mice, which are completely deficientin Tregs due to mutation in Foxp3 (the lineage regulator of Tregs), areresistant to inflammation in the pancreas, salivary glands, liver etc,although they develop multi-organ inflammation in the skin, lungs andstomach (14-16).

Deleting the IL-2 gene from the scurfy mice (the Sf.I12^(−/−) doublemutant mice) induced pancreatitis, sialoadenitis, andhepato-cholangitis, suggesting that IL-2 suppresses inflammation inthese organs independent of Tregs (14-16). However, deficiency of othersuppressive cells (such as Th2) due to loss of IL-2 may furtherexacerbate the disease.

The involvement of the organs is reminiscent of an increasing number ofpatients who have autoimmune pancreatitis (AIP). These patients alsohave parallel or subsequent inflammation in salivary glands,hepato-biliary tree, and several other organs (Khosroshahi & Stone, Aclinical overview of IgG4-related systemic disease Curr. Opin.Rheumatol., 2011, 23(1):57-66). The diseases has now been identified asa spectrum disorder, known as IgG4-related systemic disease (IgG4-RSD).These patients have elevated levels of IgG4 systemically as well as inthe inflamed organs. The production of IgG4 during autoimmunity and IgEduring allergic diseases is a result of increased TfH cell activity,which induces class-switching and somatic hypermutation in the B-cells.Our data implies that IL-2 is a negative regulator of several genesassociated with the T-follicular helper cells (TfH), which are criticalfor the maturation of B-cells and for production of high-affinityantibodies (manuscript in preparation). Recent data from other groupsalso showed that IL-2 via STATS is a negative regulator of TfH celldifferentiation (32, 33).

A strong correlation comes from Lupus patients, whose T-cells produceless IL-2, and show an increase in the TfH-mediated germinal centerformation and increased production of autoantibodies (34). Data from TID patients as well as non-obese diabetic (NOD) mouse models show strongcorrelations of hypomorphic variations in the IL-2 and IL-2 receptor(CD25) alleles (35). Similarly, in multiple sclerosis (MS), one of thestrongest genetic correlations has been linked to the hypomorphicalleles of IL-2R (36, 37). The pro-inflammatory cytokine-IFN-γ producedby Th1 cells, is increased systemically as well as locally at the sitesof inflammation in all these diseases.

Interestingly, a strong Th2 response was associated with lower incidenceand well as resolution of T1D and MS in the human patients as well asmouse models (23-26). Treatment of mice with conditions that promote theTh2 immunity was found to be beneficial in several mouse models (23,38-40).

IL-2 and Innate Lymphoid cells (ILC): A new subset of lymphocytes hasbeen identified recently by several independent groups and has beennamed ILC, Nuocytes, and natural helper cells (9, 12, 13, 41). Thesecells do not express the characteristic cell surface markers of T- andB-lymphocytes and mainly reside at the mucosal surfaces and offer firstline of defense against parasites. However, they express several cellsurface molecules, such as CD90, c-Kit and IL-7Ra, indicative of theirlymphoid origin. They also express high levels of CD25 (IL-2Ra) andT1/ST2 (IL-33R) (42-44). Stimulation of lung ILCs with IL-33 incombination with IL-2 and IL-7 resulted in production of IL-5 and IL-13,demonstrating that the ILC population in the lung resembles Type 2 ILCsthat express Th2-associated cytokines (45, 46).

IL-33, ILC, and Th2 cells: IL-33 was discovered recently and issynthesized as a 270 amino acid protein that contains a nuclearlocalization signal (NLS) at the N-terminus and a C-terminal region withstructural homology to IL-1 family cytokines (47-49). Full length IL-33localizes to the nucleus where it associates with heterochromatin andmitotic chromosomes and may function as a transcriptional repressor(47-49). The intracellular apoptosis related protease Caspase-1 cleavesIL-33 to an 18 kDa C-terminal fragment, which has structure andfunctions similar to the IL-1 family cytokines. IL-33 expression wasfound upregulated in innate immune and epithelial cells in response toparasitic infections (50, 51).

The receptor for IL-33, IL1RL1 (also known as ST2), was identified longbefore the discovery of IL-33 and was considered an orphan receptorpresent on the surface of Th2 cells until the discovery of IL-33 (52,53). IL-33 induces hetero-dimerization of IL1RL1 with IL-1RAcP, theco-receptor for IL-1 and IL-18. IL-33 is also considered an alarmin,which is released by cells undergoing apoptosis to induce clearance ofthe dying cells (52).

The ILC/Nuocytes, as well as Th2 cells, when stimulated with IL-33upregulate the expression of IL-4, IL-5, and IL-13, the criticalcytokines for the effector function of these cells against parasiticinfections. Recent studies show that IL-33 promotes the function ofILC/Nuocytes as a first line of defense, before the adaptive immunitymatures (12, 45, 54). Recent data also suggests that IL-33 production bythe dendritic cells in response to allergens may be one of themechanisms to initiate a Th2 response (55-57).

Anti-inflammatory role of IL-33: Although, the primary function of IL-33has been adjudged to boost the immunity against parasitic infections,recent data has identified several anti-inflammatory properties ofIL-33. Several of these studies indicate a skewing towards the Th2-typeof response, which results in resolution of the pro-inflammatory Th1 andTh17 responses (10, 58). Besides being chemo-attractive for andpromoting the secretion of Th2 cytokines (IL-5 and IL-13) by thedifferentiated Th2 cells, IL-33 can prime murine dendritic cells toinduce polarization of naive T cells towards a Th2 phenotype (55-57).Further, IL-33 has been shown to enhance production of IgM antibodiesand IL-5 and IL-13 production from B1 B-cells in vivo (59).

The natural IgM secreted by the B1 B-cells is widely accepted asantiinflammatory under various settings (60, 61). In experimental asthmain the ovalbumin-induced airway inflammation model, the IL-33receptor-deficient mice were not protected (62). Interestingly, inanother model adoptive transfer of IL1RL1-deficient Th2 cells intoimmuno-deficient Rag1 KO mice induced greater disease than theIL1RL1-sufficient control mice (63).

Several inflammatory diseases are driven by IL-12 and IFN-γ-induced Th1immune response and infiltration of immune cells in the target organssuch as atherosclerosis. IL-33 treatment reduced the inflammation bothin terms of lesion size as well as in terms of reduction in theinfiltrating cells in mouse models of atherosclerosis (64, 65). This wasaccompanied with a switch in the cytokine profile from INF-γ to IL-4,IL-5, and IL-13 along with an increase in the levels of protectiveanti-oxidized low-density lipoprotein (ox-LDL) IgM antibodies. On theother hand, blocking the signaling with the use of soluble ILIRLI (sST2)worsened the disease with high IFN-γ levels (66).

IL-33 has been shown to have important functions in the central nervoussystem (CNS), as indicated by strong expression of its mRNA in the brainand spinal cord and the levels are further increased under experimentalinflammatory conditions (67, 68). The microglial and astrocytes alsoexpress the IL-33 receptor as detected by flow-cytometry (69). LPSstimulation of cultured microglia and astrocytes induced the expressionof IL-33 in glial and astrocyte cultures. IL-33 treatment not onlyinduced proliferation of microglial cells, but also induced thephagocytosis and secretion of IL-10, IL-β, and TNF-a by these cells(69). Finally, a transcriptional analysis of brain tissue from patientswith Alzheimer's disease revealed that IL-33 expression was decreasedcompared to control tissues, suggesting that IL-33 may play an importantneuroprotective role during infections and inflammatory conditions (70).

IL-33/IL1RL1 axis has recently been shown to be protective in type-2diabetes (T2D). In vitro culture of adipocytes with IL-33 inducedproduction of Th2 cytokines leading to reduced lipid storage anddecreased expression of several adipogenesis related genes (71). Invivo, treatment of genetically obese diabetic mice (ob/ob) with IL-33led to protective metabolic effects with reduced adiposity, reducedfasting glucose, and improved glucose and insulin tolerance. Conversely,mice lacking ILIRLI were more susceptible to T2D upon high fat dietfeeding as compared to the controls. The protection offered by IL-33signaling was accompanied via switch in phenotype of macrophages from M1(Th1 associated pro-inflammatory) to M2 (Th2 associatedanti-inflammatory) (71). Recent studies have also shown that IL-33 caninduce the Fat-associated lymphoid cells (FALC) to secrete IL-4, IL-5and IL-13, which may be serve a protective role against inflammationduring obesity (72).

There is a long felt need in the art for compositions and methods usefulfor treating autoimmune diseases and disorders and inflammation. Thepresent application satisfies these needs.

SUMMARY OF THE INVENTION

The invention encompassed by the present application was conceived basedon the disclosure herein that the receptors for the cytokines IL-2 andIL-33 are co-expressed on subsets of immune cells that are important forboosting immunological tolerance. The present invention is useful inenhancing host immunity to parasitic infections. As disclosed herein,Treg, Th2, and the more recently discovered innate lymphoid cells,(ILC2), express high levels of the receptors for IL-2 and IL-33. Basedon the data disclosed herein, the present invention encompasses acombination therapy with IL-2 and IL-33, or a therapy with a novelfusion protein comprising active fragments of IL-2 and IL-33, tosimultaneously promote Treg and Th2 responses to offer long-termprotection against autoimmunity and inflammation by suppressing the Th1and Th17 responses, as well as inhibiting activation of several otherpro-inflammatory immune cells. The novel cytokine/fusion proteindisclosed herein has the activities of both IL-2 and IL-33 in onemolecule. This strategy is designed to increase the specificity of theIL-2 and IL-33 activities by targeting them to cell-types that aresimultaneously enriched for the receptors for both the cytokines and toincrease the binding affinity of the novel cytokine throughcooperativity. Interestingly, the ILC/Nuocytes are critically dependenton IL-2 because their numbers are drastically reduced in the IL-2deficient mice (data not presented).

The present application discloses the combination of action of twopleiotropic cytokines, IL-2 and IL-33, wherein their use as acombination results in a robust and long-lasting modulation of theimmune response to simultaneously boost multiple anti-inflammatorymechanisms, the most important being the expansion of the T-regulatorycell population, which is the major enforcer of peripheral immunetolerance. In one aspect, the combination is additive. In one aspect,the result seen with the combination is unexpectedly synergisticrelative to the effect seen when either is used alone.

Without wishing to be bound by any particular theory, it is hypothesizedherein that loss of adequate levels of IL-2 increases TfHdifferentiation and thereby induces generation of the IgG4-secretingplasma cells. Additionally, deficiency of IL-2 causes reduction in theTh2 cytokines IL-4, IL-5, and IL-13. The lack of Th2 cytokines coupledwith the increase TfH further induces the class switching of antibodiestowards more of IgG4 production. Thus, IL-2 plays important role in theorgan-specific autoimmune diseases directly by regulating Treg, byregulating Th2 response and by regulating TfH induced autoantibodyproduction.

This invention describes a novel therapeutic approach for the treatmentof autoimmune and inflammatory diseases by using a combination of twocytokines that promote anti-inflammatory responses and immune-tolerance.This therapy can be employed for boosting multiple inherent protectivemechanisms against autoimmune and inflammatory diseases. The combinationtherapy is designed to be more specific and with fewer side effects bycombining the activities in one hybrid molecule.

Interleukin (IL)-2 is a pleiotropic cytokine, which is important for thesurvival and function of several subsets of Thelper (Th) cells (1). Twoof these Th cell subsets, known as T-regulatory cells (Tregs) and Th2cells are critical for the suppression of systemic and organ-specificinflammation associated with autoimmunity and infection (2-8). Both theTregs and Th2 cells constitutively express the high-affinity receptorfor IL-2, known as IL-2Ra (CD25) and are critically dependent on IL-2for survival and function. Both T-cell subsets also express high-levelsof IL1RL1 (data not presented)—the receptor for a newly describedcytokine-IL-33, which is not only critical for the proliferation andadequate function of Th2 cells (9), but has recently been shown tosupport proliferation and function of Treg cells (10, 11). Althoughseveral cell types express CD25 and IL-33, the Treg and Th2 cells areespecially enriched for the co-expression of receptors for both of thesecytokines. Disclosed herein is the unexpected result of the synergy ofIL-2 and IL-33 to protect against autoimmune and inflammatory disordersas opposed to the use of either cytokine alone.

To better utilize the multiple effects of IL-2 and IL-33, disclosedherein is a novel fusion protein (termed IL233) that has activities ofboth IL-2 and IL-33. The IL233 fusion protein can be used to expand theexisting pool of Treg and Th2 cells to suppress inflammation mediated bythe Th1 and Th17 cells for prevention or therapy of various inflammatorydisorders that occur due to autoimmunity, transplantation, and IschemiaReperfusion Injury (IRI) and infections. In one aspect, the IL233 fusionprotein disclosed herein provides for better targeting of the activitiesof the proteins (by separating them with a linker sequence) to the Tregsand other anti-inflammatory cells. The anti-inflammatory mechanisms,thus invoked, can be used for therapy of a broad range of autoimmune andinflammatory diseases. Thus, the fusion peptide of the invention canbind to two receptors, resulting in a peptide with two separateactivities based on the interaction with two different receptors.Further disclosed herein are the unexpected results of a synergisticeffect of the combination of IL-2 and IL-33 in one fusion peptide.

The invention further encompasses different ways to join IL-2 and IL-33and their activities. For example, IL-2 and IL-33 activities could alsobe linked to each other chemically by covalent or non-covalent methods,or by co-immobilization (absorption/adsorption) on a carrier, whichcould be a cell, a liposome, a nanoparticle or matrix.

The application further discloses not just effects of these molecules invitro and mechanisms of action, but further tests the molecules in vivo.Combinations of the two cytokines and use of the novel fusion moleculedemonstrate efficacy in autoimmune type-1 diabetes, acute kidney injuryoccurring due to ischemia reperfusion, autoimmune lupusglomerulonephritis, obesity-linked Type-2 diabetes, obesity,hyperglycemia, and diabetic nephropathy.

A recently identified subset of cells known as innate lymphoid cells(ILC) or Nuocytes has been recognized as the first line of defenseagainst parasitic infections. Interestingly, the ILC/Nuocytes are alsocritically dependent on IL-2 and IL-33 for their survival and function(12, 13). The Nuocytes work in close collaboration with the Th2 cells tofight parasitic pathogens (13). One of ordinary skill in the art willappreciate that, based on the disclosure provide herein, IL233 can beused for multiple purposes, including but not limited to, boostingimmunity against parasitic infections by promoting both Nuocytes and Th2cells.

In one embodiment, the present invention provides a fusion proteinconsisting of an IL-2 fragment and an IL-33 fragment. In one aspect, theIL-2 fragment comprises residues 21-153 of human IL-2. In one aspect,the IL-33 fragment comprises residues 112-270 of human IL-33. In oneaspect, the fusion protein comprises an IL-2 and an IL-33 fragmentseparated by a short linker sequence. In one aspect, the linker sequenceis GGGGSGGGGSGGGGS (SEQ ID NO:5). The fusion protein is named IL233 andthe human version as used is disclosed below and can be referred to ashIL233. A murine version (mIL233) is also provided below.

Summary of Sequences of the Invention

Nucleic Acid Sequence for novel IL233 encoding thehuman fusion protein of SEQ ID NO: 2 SEQ ID NO: 1GAATTCGAGAACCTGTACTTCCAGGGTGCTCCGACCTCTTCTTCTACCAAGAAAACCCAGCTGCAGCTGGAACACCTGTTGCTGGACCTGCAGATGATCCTGAACGGTATCAATAACTACAAGAACCCGAAACTGACCCGTATGCTGACCTTCAAATTCTACATGCCGAAGAAAGCTACCGAACTGAAACACCTGCAGTGCCTGGAAGAGGAACTGAAACCGCTGGAAGAAGTTCTGAACCTGGCTCAGTCTAAGAACTTCCACCTGCGTCCGCGTGACCTGATCTCTAACATCAACGTTATCGTTCTGGAACTGAAAGGTTCTGAAACCACCTTCATGTGCGAATACGCTGACGAAACCGCTACCATCGTTGAGTTCCTGAACCGTTGGATCACCTTCTGCCAGTCTATCATCTCTACCCTGACCGGTGGTGGCGGTTCTGGCGGTGGCGGTTCTGGTGGCGGTGGATCCAGCATCACCGGCATCAGCCCCATCACCGAGTACCTGGCCAGCCTGAGCACCTACAACGACCAGAGCATCACCTTCGCCCTGGAGGACGAGAGCTACGAGATCTACGTGGAGGACCTGAAGAAGGACGAGAAGAAGGACAAGGTGCTGCTGAGCTACTACGAGAGCCAGCACCCCAGCAACGAGAGCGGCGACGGCGTGGACGGCAAGATGCTGATGGTGACCCTGAGCCCCACCAAGGACTTCTGGCTGCACGCCAACAACAAGGAGCACAGCGTGGAGCTGCACAAGTGCGAGAAGCCCCTGCCCGACCAGGCCTTCTTCGTGCTGCACAACATGCACAGCAACTGCGTGAGCTTCGAGTGCAAGACCGACCCCGGCGTGTTCATCGGCGTGAAGGACAACCACCTGGCCCTGATCAAGGTGGACAGCAGCGAGAACCTGTGCACCGAGAACATCCTGTTCAAGCTGAGCGAGACCTAACTCGAGamino acid sequence (308 residues) of the human IL233fusion protein (the underlined section denotes thesequence encoding the IL-2 fragment, the bold-facesequence denotes the linker segment and the remainderis the sequence encoding the IL-33 fragment) SEQ ID NO: 2GAPTSSSTKKTOLOLEHLLLDLQMILNGIYKPKLTRMLTFKFYMPKKATELKHLOCLEEELKPLEEVLNLAOSKFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCOSIISTLT GGGGSGGGGSGGGGSSITGISPITEYLASLSTYDQSITFALEDESYEIYVEDLKKDEKKDKVLLSYYESQHPSNESGDGVDGKMLMVTLSPTKDFWLHAKEHSVELHKCEKPLPDQAFFVLHNMHSNCVSFECKTDPGVFIGVKDNHLALIKVDSSENL CTENILFKLSETthe 134 amino acid residue fragment of human IL-2 used in SEQ ID NO: 2SEQ ID NO: 3 GAPTSSSTKKTOLOLEHLLLDLQMILNGIYKPKLTRMLTFKFYMPKKATELKHLOCLEEELKPLEEVLNLAQSKFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCOSIISTLTthe 159 amino acid residue fragment of human IL-33 used in SEQ ID NO: 2SEQ ID NO: 4SITGISPITEYLASLSTYDQSITFALEDESYEIYVEDLKKDEKKDKVLLSYYESQHPSNESGDGVDGKMLMVTLSPTKDFWLHAKEHSVELHKCEKPLPDQAFFVLHNMHSNCVSFECKTDPGVFIGVKDNHLALIKVDSSENLCTENILFKLSETthe 15 amino acid residue linker sequence used in SEQ ID NO: 2SEQ ID NO: 5 GGGGSGGGGSGGGGSfull length human IL-2 protein, 153 a.a., GenBank accessionnumber - P60568 SEQ ID NO: 6MYRMQLLSCIALSLALVTNSAPTSSSTKKTQLQLEHLLLDLQMILNGINYKPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLTfull length human IL-33 full length protein, 270 a.a.,GenBank accession number 095760 SEQ ID NO: 7MKPKMKYSTNKISTAKWKTASKALCFKLGKSQQKAKEVCPMYFMKLRSGLMIKKEACYFRRETTKRPSLKTGRKHKRHLVLAACQQQSTVECFAFGISGVQKYTRALHDSSITGISPITEYLASLSTYDQSITFALEDESYEIYVEDLKKDEKKDKVLLSYYESQHPSNESGDGVDGKMLMVTLSPTKDFWLHAKEHSVELHKCEKPLPDQAFFVLHNMHSNCVSFECKTDPGVFIGVKDNHLALIKVDSSENLCTENILFKLSETmurine IL-2 full length protein, 169 a.a., GenBank accessionnumber P04351 SEQ ID NO: 8MYSMQLASCVTLTLVLLVSAPTSSSTSSSTAEAQQQQQQQQQQQQHLEQLLMDLQELLSRMENYRNLKLPRMLTFKFYLPKQATELKDLQCLEDELGPLRHVLDLTQSKSFQLEDAENFISNIRVTWKLKGSDNTFECQFDDESATWDFLRRWIAFCQSIISTSPQmurine IL-33 full length protein, 266 a.a., GenBankaccession number Q8BVZ5 SEQ ID NO: 9MRPRMKYSNSKISPAKFSSTAGEALVPPCKIRRSQQKTKEFCHVYCMRLRSGLTIRKETSYFRKEPTKRYSLKSGTKHEENFSAYPRDSRKRSLLGSIQAFAASVDTLSIQGTSLLTQSPASLSTYDQSVSFVLENGCYVINVDDSGKDQEQDQVLLRYYESPCPASQSGDGVDGKKLMWMSPIKDTDIWLHANDKDYSVELQRGDVSPPEQAFFVLHKKSSDFVSFECKLPGTYIGVKDNQLALVEEKDESCIMFKLSKImurine fusion IL233 nucleic acid sequence encoding themurine IL233 fusion peptide of SEQ ID NO: 11 SEQ ID NO: 10GAATTCGAGAACCTGTACTTCCAGGGTGCACCCACTTCAAGCTCCACTTCAAGCTCTACAGCGGAAGCACAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCACCTGGAGCAGCTGTTGATGGACCTACAGGAGCTCCTGAGCAGGATGGAGAATTACAGGAACCTGAAACTCCCCAGGATGCTCACCTTCAAATTTTACTTGCCCAAGCAGGCCACAGAATTGAAAGATCTTCAGTGCCTAGAAGATGAACTTGGACCTCTGCGGCATGTTCTGGATTTGACTCAAAGCAAAAGCTTTCAATTGGAAGATGCTGAGAATTTCATCAGCAATATCAGAGTAACTGTTGTAAAACTAAAGGGCTCTGACAACACATTTGAGTGCCAATTCGATGATGAGTCAGCAACTGTGGTGGACTTTCTGAGGAGATGGATAGCCTTCTGTCAAAGCATCATCTCAACAAGCCCTCAAGGTGGTGGCGGTTCTGGCGGTGGCGGTTCTGGTGGCGGTGGATCCTCTATCCAGGGTACTTCTCTGCTGACCCAGTCTCCGGCTTCTCTGTCTACCTACAACGACCAGTCTGTTTCTTTCGTTCTGGAAAACGGTTGCTACGTTATCAACGTTGACGACTCTGGTAAAGACCAGGAACAGGACCAGGTTCTGCTGCGTTACTACGAATCTCCGTGCCCGGCTTCTCAGTCTGGTGACGGTGTTGACGGTAAGAAAGTTATGGTTAACATGTCTCCGATCAAAGACACCGACATCTGGCTGCACGCTAACGACAAAGACTACTCTGTTGAACTGCAACGTGGTGACGTTTCTCCGCCGGAACAGGCTTTCTTCGTTCTGCACAAGAAATCTTCTGACTTCGTTTCTTTCGAATGCAAGAACCTGCCGGGTACTTACATCGGTGTTAAAGACAACCAGCTCGCTCTGGTTGAAGAGAAAGACGAATCTTGCAACAACATCATGTTCAAACTGTCCAAAATCTAACTCGAGmurine IL233 (mIL233) fusion protein amino acid sequence(323 amino acid residues) SEQ ID NO: 11GAPTSSSTSSSTAEAQQQQQQQQQQQQHLEQLLMDLQELLSRMENYRNLKLPRIVILTFKFYLPKQATELKDLQCLEDELGPLRHVLDLTQSKSFQLEDAENFISNIRVTWKLKGSDNTFECQFDDESATWDFLRRWIAFCQSIISTSPQGGGGSGGGGSGGGGSSIQGTSLLTQSPASLSTYNDQSVSFVLENGCYVINVDDSGKDQEQDQVLLRYYESPCPASQSGDGVDGKKVMVMSPIKDTDIWLHANDKDYSVELQRGDVSPPEQAFFVLHKKSSDFVSFECKLPGTYIGVKDNQLALVEEKDESCIMFKLSKI

The invention provides an isolated protein comprising a IL-2/IL-33fusion molecule. Preferably, the isolated polypeptide comprising amammalian IL-2/IL-33 fusion molecule is at least about 30% homologous toa polypeptide having the amino acid sequence of at least one of SEQ IDNO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, and SEQ ID NO:11.Preferably, the isolated polypeptide is at least about 35% homologous,more preferably, about 40% homologous, more preferably, about 45%homologous, even more preferably, about 50% homologous, more preferably,about 55% homologous, preferably, about 60% homologous, more preferably,about 65% homologous, even more preferably, about 70% homologous, morepreferably, about 75% homologous, even more preferably, about 80%homologous, preferably, about 85% homologous, more preferably, about 90%homologous, even more preferably, about 95% homologous, and mostpreferably, about 99% homologous to at least one of SEQ ID NO:2, SEQ IDNO:4, SEQ ID NO:6, SEQ ID NO:8, and SEQ ID NO:11.

In one aspect, the invention provides a fusion protein comprising abiologically active domain of Interleukin-2 (IL-2) or a biologicallyactive fragment or homolog thereof, wherein the domain binds with theIL-2 receptor. Additionally, the fusion protein comprises a biologicallyactive domain of Interleukin-33 (IL-33) or a biologically activefragment or homolog thereof, wherein the IL-33 domain binds with theIL-33 receptor. In one aspect, the IL-2 domain is linked to the IL-33domain with a linker sequence. In one aspect, the linker comprises thesequence of SEQ ID NO:5 or a substantially homologous sequence.

In one aspect, the fusion protein comprises the amino acid sequence ofSEQ ID NO:2 or SEQ ID NO:1 1 or a biologically active substantiallyhomologous sequence.

In one aspect, the IL-2 portion of the fusion protein comprises thesequence of SEQ ID NO:6 or SEQ ID NO:8 and the IL-33 portion comprisesthe sequence of SEQ ID NO:7 or SEQ ID NO:9, or biologically activefragments or homologs thereof.

In one aspect, the IL-2 fragment comprises the sequence of SEQ ID NO:3,and the IL-33 fragment comprises the sequence of SEQ ID NO:4, orbiologically active fragments or homologs thereof. In one aspect, thehomolog of SEQ ID NO:3 is selected from a group comprising at least 75,80, 85, 90, and 95% homology with SEQ ID NO:3 and the homolog of SEQ IDNO:4 is selected from a group comprising at least 75, 80, 85, 90, and95% homology with SEQ ID NO:4.

In one aspect, the fusion protein is a synthetic protein.

The present invention encompasses an isolated nucleic acid encoding aprotein, wherein the protein is a fusion of active full-lengthsequences, fragments, or homologs of IL-2 and IL-33. In one aspect, theIL-2 portion or fragment is the first portion of the protein (aminoterminus). In one aspect, the IL-2 portion or fragment is second. In oneaspect, there is a linking sequence between the IL-2 portion or fragmentand the IL-33 portion or fragment. In one aspect, the protein is human.

The present invention includes an isolated nucleic acid encoding amammalian fusion IL-2/IL-33 protein molecule, or a fragment thereof,wherein the nucleic acid shares at least about 30% identity with atleast one nucleic acid having the sequence of SEQ ID NO:1 or SEQ IDNO:1 1. Preferably, the nucleic acid is at least about 35% homologous,more preferably, about 40% homologous, more preferably, about 45%homologous, even more preferably, about 50% homologous, more preferably,about 55% homologous, preferably, about 60% homologous, more preferably,about 65% homologous, even more preferably, about 70% homologous, morepreferably, about 75% homologous, even more preferably, about 80%homologous, preferably, about 85% homologous, more preferably, about 90%homologous, even more preferably, about 95% homologous, and mostpreferably, about 99% homologous to SEQ ID NO:1 or 11 disclosed herein.Even more preferably, the nucleic acid is SEQ ID NO:1 or 11.

In one embodiment, the invention provides an isolated nucleic acidcomprising a nucleic acid sequence encoding a fusion protein of theinvention. In one aspect, the invention provides an isolated nucleicacid comprising a nucleic acid sequence encoding a fusion peptide havingthe sequence of SEQ ID NOs: 2 or 1 1, or biologically active fragmentsor homologs thereof.

In one embodiment, the isolated nucleic acid comprises the sequence ofSEQ ID NOs: 1 or 10.

The present invention further provides a vector comprising an isolatednucleic acid comprising a nucleic acid sequence encoding a fusionprotein of the invention and optionally a promoter. In one aspect, thevector is selected from the group consisting of a bacterial vector, aviral vector, and a mammalian vector. In one aspect, the inventionprovides for administering the nucleic acid to a subject or to a cell.In one aspect, the isolated nucleic acid is useful for autologous celltherapy. The invention further provides a recombinant host cellcomprising an isolated nucleic acid of the invention and a recombinanthost cell comprising a vector the invention.

The present invention provides a fusion protein encoded by an isolatednucleic of the invention.

In one embodiment, the invention provides a transgenic non-human mammalcomprising an isolated nucleic acid encoding an IL-2/IL-33 fusionprotein or fragment or homolog thereof. In one aspect, the isolatednucleic acid comprises the sequence of SEQ ID NO:1 or 1 1.

In one embodiment, the present invention provides compositions andmethods for promoting/activating and stimulating proliferation of cells.In one aspect, the present invention provides compositions and methodsfor stimulating proliferation of T-regulatory (Treg), Thelper2 (Th2),and innate lymphoid cells (ILC) cells. In one aspect, the presentinvention provides compositions and methods for activating Treg, Th2 andILC cells. In one aspect, the method comprises contacting the cells withan effective amount of an IL233 fusion protein or a combination ofInterleukin-2 (IL-2) and Interleukin-33 (IL-33) proteins, orbiologically active fragments or homologs thereof. In one aspect, theinvention provides for use of a pharmaceutical composition comprising apharmaceutically acceptable carrier and an effective amount of an IL233fusion protein or a combination of Interleukin-2 (IL-2) andInterleukin-33 (IL-33) proteins, or biologically active fragments orhomologs thereof, and optionally an additional therapeutic agent,wherein the composition is used to contact cells or is administered atleast once to a subject in need thereof. In one aspect, the IL233 fusionprotein, or a biologically active homolog or fragment thereof, comprisesa fragment of IL-2 that binds to an IL-2 receptor and a fragment ofIL-33 that binds with the IL-33 receptor. In one aspect, the IL233protein comprises the amino acid sequence of SEQ ID NO:2 or SEQ IDNO:11. In one aspect, the IL-2 portion of the protein comprises thesequence of SEQ ID NO:6 or SEQ ID NO:8, and the IL-33 portion comprisesthe sequence of SEQ ID NO:7 or SEQ ID NO:9, or biologically activefragments or homologs thereof.

The present invention further provides compositions and methods fortreating diseases and disorders. In one aspect, the diseases anddisorders are autoimmune. In one aspect, inflammation is associated withthe disease or disorder being treated. In one aspect, the inventionprovides compositions and methods useful for treating a disease ordisorder including, but not limited to, diabetic nephropathy,pancreatitis, type 1 diabetes, type 2 diabetes, insulitis, lupus, lupusglomerulonephritis, obesity, acute kidney injury, renal ischemiareperfusion injury, multiple sclerosis, diabetic retinopathy, ankylosingspondylitis, autoimmune cardiomyopathy, autoimmune hemolytic anemia,autoimmune hepatitis, autoimmune lymphoproliferative syndrome,autoimmune pancreatitis, autoimmune polyendocrine syndrome, autoimmuneurticaria, autoimmune uveitis, Crohn's disease, dermatomyositis, graftversus host (GVH) disease, Hashimoto's thyroiditis, inflammatorydemyelinating diseases, interstitial cystitis, juvenile idiopathicarthritis aka Juvenile rheumatoid arthritis, lupus erythematosus,multiple sclerosis, myasthenia gravis, microscopic colitis,polymyositis, primary biliary cirrhosis, primary sclerosing cholangitis,progressive inflammatory neuropathy, rheumatoid arthritis, Sjogren'ssyndrome, systemic lupus erythematosus, transplant rejection, ulcerativecolitis (one of two types of idiopathic inflammatory bowel disease“IBD”), vasculitis, and Wegener's granulomatosis. In one aspect, themethod treats inflammation associated with a disease or disorder of theinvention.

In one embodiment, when the subject is being treated for obesity or forobesity-linked diabetes mellitus and related disorders, the methodinhibits weight gain, inhibits hyperglycemia, inhibits proteinuria, andrestores glucose tolerance.

In one embodiment, the method promotes anti-inflammatory M2 macrophages,inhibits DN-inducing M1 macrophages, and inhibits proteinuria. Inanother aspect, the method increases Treg levels in the subject.

In one embodiment, the invention provides compositions and methodsuseful for preventing or treating a disease or disorder of theinvention. In one aspect, the method prevents or treats type 1 diabetes,renal ischemia reperfusion injury, or lupus glomerulonephritis.

In one embodiment, an IL233 fusion protein or a combination ofInterleukin-2 (IL-2) and Interleukin-33 (IL-33) proteins is administeredto a subject in need thereof at a dosage ranging from about 1.0 μg/kgbody weight to about 1000 μg/kg body weight. In one aspect, the dosageis from about 10 μg/kg body weight to about 500 μg/kg body weight. Inanother aspect, the dosage is from about 20 μg/kg body weight to about100 μg/kg body weight. In a further aspect, the dosage is from about 30μg/kg body weight to about 50 μg/kg body weight. In one aspect, thedosage is 5.0, 15, 50, or 150 μg/kg of body weight. In one embodiment,the fusion protein can be administered with one or more of the cytokinesIL-2 and IL-33. One of ordinary skill in the art can determine thedosage, number of doses, and timing of doses based on the age, sex,weight, and health of the subject.

The present invention further provides compositions and methods formaking fusion peptides of the invention and for making isolated nucleicacids comprising sequences encoding the peptides.

In one aspect, a pharmaceutical composition comprising an effectiveamount of a fusion protein of the invention or a combination of IL-2 andIL-33 proteins, or fragments or homologs thereof, is administered atleast twice. In another aspect, a pharmaceutical composition isadministered at least five times. In yet another aspect, apharmaceutical composition is administered at least 10 times. One ofordinary skill in the art can determine how often to administer thecomposition based on the particular disease or disorder or how thesubject has responded to prior treatments. The present invention furtherprovides a pharmaceutical composition comprising a fusion protein of theinvention. The composition optionally comprises a pharmaceuticallyacceptable carrier. The composition may also optionally comprise anadditional therapeutic agent.

The present invention further provides kits. A kit may comprise one ormore of the fusion proteins of the invention, an isolated nucleic acidcomprising a sequence encoding a fusion protein of the invention, one ormore cytokines (IL-2 and IL-33), and a pharmaceutical compositionoptionally comprising a pharmaceutically acceptable carrier. The kit mayalso comprise one or more containers, one or more syringes, one or moreapplicators, and an instructional material for the use thereof.

Various aspects and embodiments of the invention are described infurther detail below.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawings will be provided by the Office upon request and paymentof the necessary fee.

FIGS. 1A-1C. IL-2 regulates inflammation in the Pancreas of miceindependent of Tregs. (FIG. 1A) The Foxp3 mutant scurfy (Sf) mice arecompletely deficient in Treg cell, yet they are resistant toinflammation in the pancreas despite inflammation in several otherorgans. An additional deficiency of IL-2 in Sf mice (Sf.Il2^(−/−))results in inflammation in pancreas. The IL-2 deficient mice with apartial Treg deficiency (Il2^(−/−)) also develop sever inflammation inpancreas. Islets are marked with ellipses and inflammation is shown witharrows. (FIG. 1B) The pancreas infiltrating cells produce mostly IFNγand little IL-4 as measured by intracellular staining and flow cytometryon ex vivo restimulation with PMA and Ionomicin. (FIG. 1C) Further,deletion of Th2 response genes IL-4 (Sf.Il4^(−/−)) or STATE(Sf.Stat6^(−/−)), also leads to severe pancreatitis in Treg-deficientmice. Deficiency of IFNγ (Sf.Ifrig^(−/−)) results in protection againstsuch inflammation. Islets are marked with ellipses and inflammation isshown with arrows.

FIGS. 2A and 2B. IL-2 is a negative regulator of T follicular helper(TfH) differentiation. The TfH cells produce IL-21 and promote plasmacell differentiation and production of high affinity autoantibodies.(FIG. 2A) Deficiency of IL-2 induces the expression of genes related toTfH program on CD4+ T cells. (FIG. 2B) This is accompanied withinfiltration of plasma cells (arrow) in the pancreas as measured by flowcytometry on the cells isolated from the pancreas.

FIGS. 3A and 3B. The natural Tregs (nTr) express IL-33 receptor(ILIRLI). (FIG. 3A). ILIRLI expression was measured by real-time PCR onFACS sorted natural Tregs (nTr) cells. FACS sorted naive cells (Tn) weredifferentiated in vitro into induced Tregs (iTr), effector cells (Teff),Th1, or Th2 cells and analyzed for ILIRLI expression. FACS sorted naiveT-cells (Tn) were used as a control. (FIG. 3B) Expression of ILIRLI asgated on Tregs and non-Tregs was analyzed by flow cytometry.

FIG. 4. Treatment with IL233 increases natural Tregs (Foxp3+ Helios+) inmice. Non-obese diabetic (NOD) mice were injected with 5 daily doses of1 μg equivalent (50 μg/kg of body weight) of a combination of IL-2 andIL-33 or IL233, and CD4+Foxp3+Helios+ cells were evaluated in theperipheral blood by flow cytometry. The control mice were injected withsaline. Please note that the molecular mass of IL233 is greater thanthat of IL-2 or IL-33 and is approximately the sum of the molecular massof IL-2 and IL-33. Therefore, for equimolar comparisons two-fold higherquantity of IL233 is used, e.g. for each 1 μg of IL-2 and/or IL-33, 2 μgof IL233 is used. Please note that the molecular mass of IL233 isgreater than that of IL-2 or IL-33 and is approximately the sum of themolecular mass of IL-2 and IL-33. Therefore for equimolar comparisonstwo-fold higher quantity of IL233 is used, e.g., for each 1 μg of IL-2and/or IL-33, 2 μg of IL233 is used.

FIGS. 5A-5C. Treatment of mice with IL233 protects non-obese diabetic(NOD) mice from type-1 diabetes like disease. (FIG. 5A) Female 20 weeksold NOD mice were treated once per day for 5 days with daily i.p.injections of a mixture of {circumflex over ( )}g (−50 μg/kg of bodyweight) each of IL-2 and IL-33 (orange) or IL233 (green; molarequivalent to 1 μg IL-2) before the onset of hyperglycemia. Thetreatment with the cytokines protected the mice for long-term againstonset of type-1 diabetes like disease as compared to untreated controls(blue). (FIG. 5B) NOD females that were early diabetic (blood glucose150+10 mg/dl) were treated once for 5 days with daily i.p. injections of{circumflex over ( )}g of recombinant IL-2 (red) or molar equivalent ofIL233 (green). Treatment with IL233, but not IL-2 alone protected themice from ongoing hyperglycemia as shown by the delay in the diseasekinetics with 2/5 mice showing complete protection. Blue arrow indicatesstart of the treatment. (FIG. 5C) In another experiment, NOD mice withearly diabetes (blood glucose of 150+10 mg/dL) were treated with onetime with 5-daily injections of 0.3 μg/mouse (15 μg/kg of body weight)of IL-2 alone, IL-33 alone, combination of IL-2 and IL-33 or molarequivalent of IL233. The control mice were injected with saline only.The numbers in parenthesis on the right represent the number of mice ineach group. Please note that the molecular mass of IL233 is greater thanthat of IL-2 or IL-33 and is approximately the sum of the molecular massof IL-2 and IL-33. Therefore for equimolar comparisons two-fold higherquantity of IL233 is used, e.g., for each 1 μg of IL-2 and/or IL-33, 2μg of IL233 is used.

FIG. 6. Treatment with IL233 Expands Treg Cells Especially in thePancreatic Lymph Nodes of NOD Mice.

Non-obese diabetic mice with early diabetes (blood glucose 150+_10mg/dL) were injected daily for five (5.0) consecutive days with 1.0 μgIL-2 or IL-33 or combination of IL-2 and IL-33 or molar equivalent ofIL233. The animals were followed over time. The control mice wereinjected with saline. Six weeks post-treatment, the mice that recoveredfrom hyperglycemia or the mice that were severely diabetic (bloodglucose of 600 mg/dL for two consecutive days) were euthanized and thespleen and pancreatic lymph nodes were analyzed for the CD4+Foxp3+Tregcells. The numbers in the parenthesis show the number of mice analyzed.Please note that the molecular mass of IL233 is greater than that ofIL-2 or IL-33 and is approximately the sum of the molecular masses ofIL-2 and IL-33. Therefore, for equimolar comparisons two-fold higherquantity of IL233 is used, e.g. for each 1 μg of IL-2 and/or IL-33, 2 μgof IL233 is used.

FIGS. 7A-7F. Treatment with IL233 or mixture of IL-2 and IL-33 protectsC57BL/6 mice from renal ischemia reperfusion injury (IRI). C57BL/6 micewere treated daily for 5 consecutive days with doses of the indicatedamounts (0.3, 1.0, or 3.0 μg per animal, i.e., 15, 50, or 150 μg/kg ofbody weight) of a combination of IL-2 and IL-33 or with molarequivalents (0.1, 0.3 or 1.0 i.e., 5, 15, or 50 μg/kg) IL233 fusioncytokine starting at a one third lower amount than the combination ofIL-2 and IL-33. On day 7, the renal pedicle was clamped for 26 minutesfollowed by 18 hours reperfusion. High plasma creatinine levels indicateloss of kidney function. The IL233 fusion was 3-fold more effective thanthe mixture of IL-2 and IL-33 in protecting mice against loss of renalfunction and inflammation as measure by Plasma creatinine levels (FIG.7A) or Histological score. (FIG. 7B). The protection was accompaniedwith reduction of the activated CD4+ T cells (FIG. 7C), CD8+ T-cells(not shown) and activated B-cells (FIG. 7D) in the spleen. TNFaproducing CD4 (FIG. 7E) and CD8 (FIG. 7F) T-cells and IFN-γ producingcells (not shown) were also reduced by the cytokine treatment asmeasured by flow cytometry of the cells isolated from the spleen. Pleasenote that the molecular mass of IL233 is greater than that of IL-2 orIL-33 and is approximately the sum of the molecular mass of IL-2 andIL-33. Therefore for equimolar comparisons two-fold higher quantity ofIL233 is used, e.g. for each 1 μg of IL-2 and/or IL-33, 2 μg of IL233 isused.

FIGS. 8A and 8B. Treatment with IL233 protects lupus prone NZM2328 micefrom lupus glomerulonephritis (GN). Three-month-old NZM2328 female mice(5 mice per group) were injected with recombinant IL-2 or IL233 fusionprotein ({circumflex over ( )}gIL-2 equivalent of each) daily for 5 days(green arrows). The control mice received saline only. On day 12 all themice with injected with Ad-IFNa (adenovirus expressing IFNa) toaccelerate the lupus nephritis (red double-headed arrow). The mice weremonitored periodically for kidney function (proteinuria with dip stick)and mortality. (FIG. 8A) The peripheral blood of mice was analyzed on dland d9 of the recombinant cytokine treatment. Treatment with both IL-2and IL233 resulted in an increase in the Treg levels, as measured byflow cytometry. Treatment with IL233, but not IL-2 resulted in adecrease in the production of TNF-a (a pro-inflammatory cytokine knownto contribute to lupus GN) as measured by intracellular staining oflymphocytes for TNF-a after a 5-hour ex-vivo stimulation with Phorbolmyristate acetate and Ionomycin. Individual mice are shown. (FIG. 8B)Proteinuria was measure by dipstick and mice with a “+++” score on thedipstick were considered to have severe proteinuria (top). The data ispresented as “percent proteinuria free”. The control mice (Blue)developed severe proteinuria rapidly, while treatment with IL-2 (red)protected partially. The mice treated with IL233 (green) were completelyprotected against severe proteinuria at the termination of theexperiment. IL233 treatment offered complete protection againstmortality, which IL-2 treatment protected partially (bottom). Pleasenote that the molecular mass of IL233 is greater than that of IL-2 orIL-33 and is approximately the sum of the molecular mass of IL-2 andIL-33. Therefore for equimolar comparisons two-fold higher quantity ofIL233 is used, e.g. for each 1 μg of IL-2 and/or IL-33, 2 μg of IL233 isused.

FIGS. 9A-9H. Treatment with IL233 as well as the IL-2 and IL-33combination is more effective than either cytokine alone to protectlupus prone NZM2328 mice from GN. In another similar experiment, 3-monthold NZM2328 mice were injected for five consecutive days (green arrow)with {circumflex over ( )}g equivalent daily of IL-2 or IL-33 or amixture of IL-2 and IL-33 or IL233. Three days later mice were injectedwith Adenovirus expressing IFNa to accelerate lupus GN. (FIG. 9A)Combined treatment with IL-2 and IL-33, especially as a IL233 cytokineprotected NZM2328 mice from severe proteinuria (top) and mortality(bottom) in IFNa-induced accelerated GN (green arrows-cytokinetreatment; red arrows IFNa; n=5). (FIG. 9B) Representative H & E stainedkidney sections show enlarged glomeruli (highlighted by black-dottedcircle; higher magnification in the inset), mesangial expansion,glomerulosclerosis (inset) and leukocytic infiltration in the controlmice, but not in the IL233-treatment group (quantified in FIG. 9C).(FIG. 9D) IL233 treatment, although inhibited glomerular hypertrophy,did not significantly alter Complement C3 and total IgG immune complexdeposition. (FIG. 9E) IL233 treatment skewed the circulating anti-dsDNAantibodies from IgG2a to IgG2b, p<0.01; n=3. IL233 treatment increasedFoxp3+ Tregs as measured in the lymph node (LN) of mice (FIG. 9F)leading to lower ratios of Tregs to IFNy+ (FIG. 9G) & TNFa+ (FIG. 9H)CD4 T-cells when analyzed 12-wks post initial treatment or when thecontrol of IL-2 or IL-33 treated mice were moribund. Please note thatthe molecular mass of IL233 is greater than that of IL-2 or IL-33 and isapproximately the sum of the molecular mass of IL-2 and IL-33. Thereforefor equimolar comparisons two-fold higher quantity of IL233 is used,e.g. for each 1 μg of IL-2 and/or IL-33, 2 μg of IL233 is used.“♦”=individual mice; bar=mean.

FIGS. 10A-10E. Treatment with IL233 inhibits progression of obesity,type-2 diabetes (T2D; also referred to as diabetes mellitus type 2), anddiabetic nephropathy in mice genetically predisposed for obesity. Fiveto six weeks old BTBR.ob/ob (Ob-obese mice, due to mutation in Leptingene) or BTBR.ob/+ (Het, non-obese mice) were treated once with 5-dailydoses of 50 μg/kg of IL233 or saline (green arrows). The mice weremonitored for CD4 Foxp3 Tregs (FIG. 10A), body weight (FIG. 10B), bloodglucose (FIG. 10C), proteinuria (FIG. 10D). The glucose tolerance of themice treated with IL233 also improved to near non-obese levels (FIG.10E). As shown below, IL233 treatment increased the Treg levels andinhibited the weight gain, hyperglycemia, and proteinuria, and restoredglucose tolerance.

FIGS. 11A-11D. The nucleotide sequence (SEQ ID NO:1) of the syntheticgene (FIG. 11A) and the amino acid sequence (SEQ ID NO:2) (FIG. 11B) ofthe human recombinant IL233 fusion protein are depicted. The Blue colordenotes the IL-2 coding sequence, the red color is the IL-33 encodingsequence, the black residues are the linker segments to provideflexibility to the fusion protein and the yellow highlighted part codesfor the TEV protease cleave site. The major restriction sites areunderlined in A. (FIG. 11C) mouse IL233 fusion gene nucleic acidsequence (SEQ ID NO:10). (FIG. 11D) mouse IL233 fusion protein sequence(SEQ ID NO:1 1).

FIGS. 12A-12D. Schematic illustration of the different modes of actionof the IL233 fusion protein. (FIG. 12A) Schematic representation of theIL233 fusion protein and its receptors. IL233 may bind to two receptorson the same cell (FIG. 12B) or on adjacent cells bearing the receptorsfor IL-2 and IL-33 (FIG. 12C). In a multimeric complex of cells, IL233binding to Tregs, Th2, or ILC2 on one end and to antigen presenting cell(DC or macrophages) on the other end may induce tolerance, thussuppressing Th1, TH17, or TfH activation (see FIG. 12D).

FIG. 13. Schematic illustration of a proposed model of usingcombinations of IL-2 and IL-33 or a fusion protein of IL-2 and IL-33.Without wishing to be bound by any particular theory, it washypothesized herein that, owing to the expression of the receptors forIL-2 and IL-33 on the Tregs, Th2 cells and Innate Lymphoid cells type 2(ILC2), combining the activities of IL-2 and IL-33 could invoke multiplemechanisms for suppression of autoimmune and inflammatory diseases,which include (a) activation and recruitment of Tregs/ILC2 forperipheral tolerance (b) skewing of the immune response towards Th2 forsuppression of the pro-inflammatory Th1 and Th17 cells (c) inhibitingthe T-follicular helper (TfH) cells, which induce high-affinityautoantibodies and (d) induce tolerogenic alternately activatedmacrophages. Owing to the high-level constitutive expression of thereceptors for both IL-2 and IL-33, a treatment with combination of IL-2and IL-33 will increase the targeting of Tregs, Th2 and ILC2.

DETAILED DESCRIPTION

Abbreviations and Acronyms

AAM—alternately activated macrophages

AGE—advanced glycation end-product

AIP—autoimmune pancreatitis

AKI—acute kidney injury

AMDCC—Animal Models of Diabetic Complications Consortium

APC—antigen presenting cell

DM—diabetes mellitus (can be type 1 or 2)

DN—diabetic nephropathy

ESRD—end-stage renal disease

FALC—fat-associated lymphoid cell

GN—glomerulonephritis

GVHD—graft versus host disease

hIL233—the human fusion peptide of IL-2 and IL-33 (SEQ ID NO:2)

ILIRLI—IL-33 receptor (also known as ST2)

IL233—a fusion peptide of IL-2 and IL-33

IL-2/IL-33—a fusion peptide comprising active fragments of IL-2 andIL-33

IL-33—interleukin 33

ILC—innate lymphoid cells, also referred to as nuocytes

IL-2—interleukin 2

IRI—ischemic reperfusion injury

iTr—induced Treg

g/kg body wt. —grams per kilogram body weight

LB—Luria Bertani

μg/kg body wt. —micrograms per kilogram body weight

M1 macrophage—Th1 associated pro-inflammatory macrophage

M2 macrophage—Th2 associated anti-inflammatory macrophage m2—murine IL-2protein

m2—murine IL-2 protein

m33—murine IL-33 protein

m233—murine IL233 fusion cytokine, also referred to as mIL233

mIL233—murine fusion protein of murine IL-2 and IL-33 (SEQ ID NO:11)

mL233—the human fusion peptide of IL-2 and IL-33 (SEQ ID NO:2)

MS—multiple sclerosis

NLS—nuclear localization signal

NOD—non-obese diabetic

nTr—natural Treg

r—recombinant

RLN—renal lymph node

RSD—related systemic disease

T1D—type 1 diabetes me llitus

T2D—type 2 diabetes mellitus

TAC—transverse aortic constriction

Tfh—T-follicular helper cell

Th—T helper cell

Th1—Thelper1 cell

Th2—Thelper2 cell

Th17—Thelper17 cell

Tn—naive T cell

Treg—T regulatory

UAER—urinary albumin excretion rate

Definitions

In describing and claiming the invention, the following terminology willbe used in accordance with the definitions set forth below.

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e., to at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or more thanone element.

The term “about,” as used herein, means approximately, in the region of,roughly, or around. When the term “about” is used in conjunction with anumerical range, it modifies that range by extending the boundariesabove and below the numerical values set forth. In general, the term“about” is used herein to modify a numerical value above and below thestated value by a variance of 10%. Therefore, about 50% means in therange of 45%-55%. Numerical ranges recited herein by endpoints includeall numbers and fractions subsumed within that range (e.g. 1 to 5includes 1, 1.5, 2, 2.75, 3, 3.90, 4, and 5). It is also to beunderstood that all numbers and fractions thereof are presumed to bemodified by the term “about.”

The terms “additional therapeutically active compound” or “additionaltherapeutic agent”, as used in the context of the present invention,refers to the use or administration of a compound for an additionaltherapeutic use for a particular injury, disease, or disorder beingtreated. Such a compound, for example, could include one being used totreat an unrelated disease or disorder, or a disease or disorder whichmay not be responsive to the primary treatment for the injury, diseaseor disorder being treated.

As use herein, the terms “administration of” and or “administering” acompound should be understood to mean providing a compound of theinvention or a prodrug of a compound of the invention to a subject inneed of treatment.

The term “adult” as used herein, is meant to refer to any non-embryonicor non-juvenile subject. For example the term “adult adipose tissue stemcell,” refers to an adipose stem cell, other than that obtained from anembryo or juvenile subject.

Cells or tissue are “affected” by an injury, disease or disorder if thecells or tissue have an altered phenotype relative to the same cells ortissue in a subject not afflicted with the injury, disease, condition,or disorder.

As used herein, an “agonist” is a composition of matter that, whenadministered to a mammal such as a human, enhances or extends abiological activity of interest. Such effect may be direct or indirect.

A disease, condition, or disorder is “alleviated” if the severity of asymptom of the disease or disorder, the frequency with which such asymptom is experienced by a patient, or both, are reduced.

As used herein, “alleviating an injury, disease or disorder symptom,”means reducing the frequency or severity of the symptom. As used herein,amino acids are represented by the full name thereof, by the threeletter code corresponding thereto, or by the one-letter codecorresponding thereto, as indicated in the following table:

Three-Letter One- Full Name Code Letter Aspartic Acid Asp D GlutamicAcid Glu E Lysine Lys K Arginine Arg R Histidine His H Tyrosine Tyr YCysteine Cys C Asparagine Asn N Glutamine Gin Q Serine Ser S ThreonineThr T Glycine Gly G Alanine Ala A Valine Val V Leucine Leu L IsoleucineHe I Methionine Met M Proline Pro P Phenylalanine Phe F Tryptophan Trp W

The term “amino acid” as used herein is meant to include both naturaland synthetic amino acids, and both D and L amino acids. “Standard aminoacid” means any of the twenty standard L-amino acids commonly found innaturally occurring peptides. “Nonstandard amino acid residue” means anyamino acid, other than the standard amino acids, regardless of whetherit is prepared synthetically or derived from a natural source. As usedherein, “synthetic amino acid” also encompasses chemically modifiedamino acids, including but not limited to salts, amino acid derivatives(such as amides), and substitutions. Amino acids contained within thepeptides of the present invention, and particularly at the carboxy- oramino-terminus, can be modified by methylation, amidation, acetylationor substitution with other chemical groups which can change thepeptide's circulating half-life without adversely affecting theiractivity. Additionally, a disulfide linkage may be present or absent inthe peptides of the invention.

The term “amino acid” is used interchangeably with “amino acid residue,”and may refer to a free amino acid and to an amino acid residue of apeptide. It will be apparent from the context in which the term is usedwhether it refers to a free amino acid or a residue of a peptide.

Amino acids have the following general structure:

Amino acids may be classified into seven groups on the basis of the sidechain R: (1) aliphatic side chains, (2) side chains containing a hydroxylic (OH) group, (3) side chains containing sulfur atoms, (4) sidechains containing an acidic or amide group, (5) side chains containing abasic group, (6) side chains containing an aromatic ring, and (7)proline, an imino acid in which the side chain is fused to the aminogroup.

The nomenclature used to describe the peptide compounds of the presentinvention follows the conventional practice wherein the amino group ispresented to the left and the carboxy group to the right of each aminoacid residue. In the formulae representing selected specific embodimentsof the present invention, the amino- and carboxy-terminal groups,although not specifically shown, will be understood to be in the formthey would assume at physiologic pH values, unless otherwise specified.

The term “basic” or “positively charged” amino acid as used herein,refers to amino acids in which the R groups have a net positive chargeat pH 7.0, and include, but are not limited to, the standard amino acidslysine, arginine, and histidine.

As used herein, an “analog” of a chemical compound is a compound that,by way of example, resembles another in structure but is not necessarilyan isomer (e.g., 5-fluorouracil is an analog of thymine).

An “antagonist” is a composition of matter that when administered to amammal such as a human, inhibits or impedes a biological activityattributable to the level or presence of an endogenous compound in themammal. Such effect may be direct or indirect.

The term “antibody,” as used herein, refers to an immunoglobulinmolecule which is able to specifically bind to a specific epitope on anantigen. Antibodies can be intact immunoglobulins derived from naturalsources or from recombinant sources and can be immunoreactive portionsof intact immunoglobulins. Antibodies are typically tetramers ofimmunoglobulin molecules. The antibodies in the present invention mayexist in a variety of forms including, for example, polyclonalantibodies, monoclonal antibodies, Fv, Fab and F(ab)₂, as well as singlechain antibodies and humanized antibodies.

The term “antibody,” as used herein, refers to an immunoglobulinmolecule which is able to specifically bind to a specific epitope on anantigen. Antibodies can be intact immunoglobulins derived from naturalsources or from recombinant sources and can be immunoreactive portionsof intact immunoglobulins. Antibodies are typically tetramers ofimmunoglobulin molecules. The antibodies in the present invention mayexist in a variety of forms including, for example, polyclonalantibodies, monoclonal antibodies, Fv, Fab and F(ab)2, as well as singlechain antibodies and humanized antibodies (Harlow et al., 1999, UsingAntibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press,NY; Harlow et al., 1989, Antibodies: A Laboratory Manual, Cold SpringHarbor, N.Y.; Houston et al, 1988, Proc. Natl. Acad. Sci. USA85:5879-5883; Bird et al, 1988, Science 242:423-426).

An “antibody heavy chain,” as used herein, refers to the larger of thetwo types of polypeptide chains present in all antibody molecules.

An “antibody light chain,” as used herein, refers to the smaller of thetwo types of polypeptide chains present in all antibody molecules.

By the term “synthetic antibody” as used herein, is meant an antibodywhich is generated using recombinant DNA technology, such as, forexample, an antibody expressed by a bacteriophage as described herein.The term should also be construed to mean an antibody which has beengenerated by the synthesis of a DNA molecule encoding the antibody andwhich DNA molecule expresses an antibody protein, or an amino acidsequence specifying the antibody, wherein the DNA or amino acid sequencehas been obtained using synthetic DNA or amino acid sequence technologywhich is available and well known in the art.

The term “antigen” as used herein is defined as a molecule that provokesan immune response. This immune response may involve either antibodyproduction, or the activation of specific immunologically-competentcells, or both. An antigen can be derived from organisms, subunits ofproteins/antigens, killed or inactivated whole cells or lysates.

A ligand or a receptor (e.g., an antibody) “specifically binds to” or“is specifically immunoreactive with” a compound when the ligand orreceptor functions in a binding reaction which is determinative of thepresence of the compound in a sample of heterogeneous compounds. Thus,under designated assay (e.g., immunoassay) conditions, the ligand orreceptor binds preferentially to a particular compound and does not bindin a significant amount to other compounds present in the sample. Forexample, a polynucleotide specifically binds under hybridizationconditions to a compound polynucleotide comprising a complementarysequence; an antibody specifically binds under immunoassay conditions toan antigen bearing an epitope against which the antibody was raised. Avariety of immunoassay formats may be used to select antibodiesspecifically immunoreactive with a particular protein. For example,solid-phase ELISA immunoassays are routinely used to select monoclonalantibodies specifically immunoreactive with a protein. See Harlow andLane (1988, Antibodies, A Laboratory Manual, Cold Spring HarborPublications, New York) for a description of immunoassay formats andconditions that can be used to determine specific immunoreactivity.

The term “antimicrobial agents” as used herein refers to anynaturally-occurring, synthetic, or semi-synthetic compound orcomposition or mixture thereof, which is safe for human or animal use aspracticed in the methods of this invention, and is effective in killingor substantially inhibiting the growth of microbes. “Antimicrobial” asused herein, includes antibacterial, antifungal, and antiviral agents.

As used herein, the term “antisense oligonucleotide” or antisensenucleic acid means a nucleic acid polymer, at least a portion of whichis complementary to a nucleic acid which is present in a normal cell orin an affected cell. “Antisense” refers particularly to the nucleic acidsequence of the non-coding strand of a double stranded DNA moleculeencoding a protein, or to a sequence which is substantially homologousto the non-coding strand. As defined herein, an antisense sequence iscomplementary to the sequence of a double stranded DNA molecule encodinga protein. It is not necessary that the antisense sequence becomplementary solely to the coding portion of the coding strand of theDNA molecule. The antisense sequence may be complementary to regulatorysequences specified on the coding strand of a DNA molecule encoding aprotein, which regulatory sequences control expression of the codingsequences. The antisense oligonucleotides of the invention include, butare not limited to, phosphorothioate oligonucleotides and othermodifications of oligonucleotides.

The term “associated with ischemia” as used herein means that an injury,disease, or disorder that is being treated or which is being preventedeither develops as a result of ischemia or ischemia develops as a resultof the injury disease or disorder, i.e., the two are closely linked.

The term “binding” refers to the adherence of molecules to one another,such as, but not limited to, enzymes to substrates, ligands toreceptors, antibodies to antigens, DNA binding domains of proteins toDNA, and DNA or RNA strands to complementary strands.

“Binding partner,” as used herein, refers to a molecule capable ofbinding to another molecule.

The term “biocompatible”, as used herein, refers to a material that doesnot elicit a substantial detrimental response in the host.

As used herein, the term “biologically active fragments” or “bioactivefragment” of the polypeptides encompasses natural or synthetic portionsof the full-length protein that are capable of specific binding to theirnatural ligand or of performing the function of the protein.

The term “biological sample,” as used herein, refers to samples obtainedfrom a living organism, including skin, hair, tissue, blood, plasma,cells, sweat, and urine.

As used herein, the term “biologically active fragments” or “bioactivefragment” of the polypeptides encompasses natural or synthetic portionsof the full-length protein that are capable of specific binding to theirnatural ligand or of performing the function of the protein.

A “biomarker” is a specific biochemical in the body which has aparticular molecular feature that makes it useful for measuring theprogress of disease or the effects of treatment, or for measuring aprocess of interest.

As used herein, the term “carrier molecule” refers to any molecule thatis chemically conjugated to the antigen of interest that enables animmune response resulting in antibodies specific to the native antigen.

As used herein, the term “chemically conjugated,” or “conjugatingchemically” refers to linking the antigen to the carrier molecule. Thislinking can occur on the genetic level using recombinant technology,wherein a hybrid protein may be produced containing the amino acidsequences, or portions thereof, of both the antigen and the carriermolecule. This hybrid protein is produced by an oligonucleotide sequenceencoding both the antigen and the carrier molecule, or portions thereof.This linking also includes covalent bonds created between the antigenand the carrier protein using other chemical reactions, such as, but notlimited to glutaraldehyde reactions. Covalent bonds may also be createdusing a third molecule bridging the antigen to the carrier molecule.These cross-linkers are able to react with groups, such as but notlimited to, primary amines, sulfhydryls, carbonyls, carbohydrates orcarboxylic acids, on the antigen and the carrier molecule. Chemicalconjugation also includes non-covalent linkage between the antigen andthe carrier molecule.

A “coding region” of a gene consists of the nucleotide residues of thecoding strand of the gene and the nucleotides of the non-coding strandof the gene which are homologous with or complementary to, respectively,the coding region of an mR A molecule which is produced by transcriptionof the gene.

The term “competitive sequence” refers to a peptide or a modification,fragment, derivative, or homolog thereof that competes with anotherpeptide for its cognate binding site.

“Complementary” refers to the broad concept of sequence complementaritybetween regions of two nucleic acid strands or between two regions ofthe same nucleic acid strand. It is known that an adenine residue of afirst nucleic acid region is capable of forming specific hydrogen bonds(“base pairing”) with a residue of a second nucleic acid region which isantiparallel to the first region if the residue is thymine or uracil. Asused herein, the terms “complementary” or “complementarity” are used inreference to polynucleotides (i.e., a sequence of nucleotides) relatedby the base-pairing rules. For example, for the sequence “A-G-T,” iscomplementary to the sequence “T-C-A.”

Similarly, it is known that a cytosine residue of a first nucleic acidstrand is capable of base pairing with a residue of a second nucleicacid strand which is antiparallel to the first strand if the residue isguanine. A first region of a nucleic acid is complementary to a secondregion of the same or a different nucleic acid if, when the two regionsare arranged in an antiparallel fashion, at least one nucleotide residueof the first region is capable of base pairing with a residue of thesecond region. Preferably, the first region comprises a first portionand the second region comprises a second portion, whereby, when thefirst and second portions are arranged in an antiparallel fashion, atleast about 50%, and preferably at least about 75%, at least about 90%,or at least about 95% of the nucleotide residues of the first portionare capable of base pairing with nucleotide residues in the secondportion. More preferably, all nucleotide residues of the first portionare capable of base pairing with nucleotide residues in the secondportion.

The term “complex”, as used herein in reference to proteins, refers tobinding or interaction of two or more proteins. Complex formation orinteraction can include such things as binding, changes in tertiarystructure, and modification of one protein by another, such asphosphorylation.

A “compound,” as used herein, refers to any type of substance or agentthat is commonly considered a drug, or a candidate for use as a drug, aswell as combinations and mixtures of the above. As used herein, the term“conservative amino acid substitution” is defined herein as an aminoacid exchange within one of the following five groups:

I. Small aliphatic, nonpolar or slightly polar residues:

-   -   Ala, Ser, Thr, Pro, Gly;

II. Polar, negatively charged residues and their amides:

-   -   Asp, Asn, Glu, Gin;

III. Polar, positively charged residues:

-   -   His, Arg, Lys;

IV. Large, aliphatic, nonpolar residues:

-   -   Met Leu, He, Val, Cys

V. Large, aromatic residues:

-   -   Phe, Tyr, Trp

A “control” cell, tissue, sample, or subject is a cell, tissue, sample,or subject of the same type as a test cell, tissue, sample, or subject.The control may, for example, be examined at precisely or nearly thesame time the test cell, tissue, sample, or subject is examined. Thecontrol may also, for example, be examined at a time distant from thetime at which the test cell, tissue, sample, or subject is examined, andthe results of the examination of the control may be recorded so thatthe recorded results may be compared with results obtained byexamination of a test cell, tissue, sample, or subject. The control mayalso be obtained from another source or similar source other than thetest group or a test subject, where the test sample is obtained from asubject suspected of having a disease or disorder for which the test isbeing performed.

A “test” cell, tissue, sample, or subject is one being examined ortreated.

“Cytokine,” as used herein, refers to intercellular signaling molecules,the best known of which are involved in the regulation of mammaliansomatic cells. A number of families of cytokines, both growth promotingand growth inhibitory in their effects, have been characterizedincluding, for example, interleukins, interferons, chemokines, proteinor peptide hormones, and transforming growth factors. A number of othercytokines are known to those of skill in the art. The sources,characteristics, targets and effector activities of these cytokines havebeen described.

The term “delivery vehicle” refers to any kind of device or materialwhich can be used to deliver compounds in vivo or can be added to acomposition comprising compounds administered to a plant or animal. Thisincludes, but is not limited to, implantable devices, aggregates ofcells, matrix materials, gels, nucleic acids, etc.

As used herein, a “derivative” of a compound, when referring to achemical compound, is one that may be produced from another compound ofsimilar structure in one or more steps, as in replacement of H by analkyl, acyl, or amino group.

A “derivative protein or peptide,” as used herein, includes any proteinor peptide, which in its entirety, or in part, comprises a substantiallysimilar amino acid sequence to an IL-2 and IL-33 fusion peptide (IL233)and has IL233 biological activity as disclosed herein. Derivatives ofIL233 may be characterized by single or multiple amino acidsubstitutions, deletions, additions, or replacements. These derivativesmay include (a) derivatives in which one or more amino acid residues aresubstituted with conservative or non-conservative amino acids; (b)derivatives in which one or more amino acids are added to one or more ofthe components of the fusion peptide (c) derivatives in which one ormore of the amino acids includes a substituent group; (d) derivatives inwhich one of the constituent groups or a portion thereof is fused toanother peptide (e.g., serum albumin or protein transduction domain);(e) derivatives in which one or more nonstandard amino acid residues(i.e., those other than the 20 standard L-amino acids found in naturallyoccurring proteins) are incorporated or substituted into one of the IL-2or IL-33 substituents; and (f) derivatives in which one or more nonaminoacid linking groups are incorporated into or replace a portion of one ofthe portions of the fusion protein. A derivative protein may also bereferred to as homologous when used in the context described herein. Aderivative or homolog as described or claimed herein will have similaractivity to IL-2, IL-33, and the fusion peptide IL233 as disclosedherein.

The use of the word “detect” and its grammatical variants refers tomeasurement of the species without quantification, whereas use of theword “determine” or “measure” with their grammatical variants are meantto refer to measurement of the species with quantification. The terms“detect” and “identify” are used interchangeably herein.

As used herein, a “detectable marker” or a “reporter molecule” is anatom or a molecule that permits the specific detection of a compoundcomprising the marker in the presence of similar compounds without amarker. Detectable markers or reporter molecules include, e.g.,radioactive isotopes, antigenic determinants, enzymes, nucleic acidsavailable for hybridization, chromophores, fluorophores,chemiluminescent molecules, electrochemically detectable molecules, andmolecules that provide for altered fluorescence-polarization or alteredlight-scattering.

As used herein, the term “diagnosis” refers to detecting a disease ordisorder or a risk or propensity for development of a disease ordisorder, for the types of diseases or disorders encompassed by theinvention. In any method of diagnosis there exist false positives andfalse negatives. Any one method of diagnosis does not provide 100%accuracy.

A “disease” is a state of health of an animal wherein the subject cannotmaintain homeostasis, and wherein if the disease is not ameliorated thenthe subject's health continues to deteriorate. In contrast, a “disorder”in an subject is a state of health in which the animal is able tomaintain homeostasis, but in which the subject's state of health is lessfavorable than it would be in the absence of the disorder. Leftuntreated, a disorder does not necessarily cause a further decrease inthe subject's state of health.

As used herein, the term “domain” refers to a part of a molecule orstructure that shares common physicochemical features, such as, but notlimited to, hydrophobic, polar, globular and helical domains orproperties such as ligand binding, signal transduction, cell penetrationand the like. Specific examples of binding domains include, but are notlimited to, DNA binding domains and ATP binding domains. As used herein,the term “effector domain” refers to a domain capable of directlyinteracting with an effector molecule, chemical, or structure in thecytoplasm which is capable of regulating a biochemical pathway.

The term “downstream” when used in reference to a direction along anucleotide sequence means the 5′ to 3′ direction. Similarly, the term“upstream” means the 3′ to 5′ direction.

As used herein, an “effective amount” means an amount sufficient toproduce a selected effect, such as alleviating symptoms of a disease ordisorder. In the context of administering compounds in the form of acombination, such as multiple compounds, the amount of each compound,when administered in combination with another compound(s), may bedifferent from when that compound is administered alone. Thus, aneffective amount of a combination of compounds refers collectively tothe combination as a whole, although the actual amounts of each compoundmay vary. The term “more effective” means that the selected effect isalleviated to a greater extent by one treatment relative to the secondtreatment to which it is being compared.

“Encoding” refers to the inherent property of specific sequences ofnucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, toserve as templates for synthesis of other polymers and macromolecules inbiological processes having either a defined sequence of nucleotides(i.e., rRNA, tR A and mRNA) or a defined sequence of amino acids and thebiological properties resulting therefrom. Thus, a gene encodes aprotein if transcription and translation of mRNA corresponding to thatgene produces the protein in a cell or other biological system. Both thecoding strand, the nucleotide sequence of which is identical to the mRNAsequence and is usually provided in sequence listings, and thenon-coding strand, used as the template for transcription of a gene orcDNA, can be referred to as encoding the protein or other product ofthat gene or cDNA.

Unless otherwise specified, a “nucleotide sequence encoding an aminoacid sequence” includes all nucleotide sequences that are degenerateversions of each other and that encode the same amino acid sequence.Nucleotide sequences that encode proteins and RNA may include introns.

An “enhancer” is a DNA regulatory element that can increase theefficiency of transcription, regardless of the distance or orientationof the enhancer relative to the start site of transcription.

As used herein, an “essentially pure” preparation of a particularprotein or peptide is a preparation wherein at least about 95%, andpreferably at least about 99%, by weight, of the protein or peptide inthe preparation is the particular protein or peptide.

As used in the specification and the appended claims, the terms “forexample,” “for instance,” “such as,” “including” and the like are meantto introduce examples that further clarify more general subject matter.Unless otherwise specified, these examples are provided only as an aidfor understanding the invention, and are not meant to be limiting in anyfashion.

The terms “formula” and “structure” are used interchangeably herein.

As used herein the term “expression” when used in reference to a gene orprotein, without further modification, is intended to encompasstranscription of a gene and/or translation of the transcript into aprotein.

A “fragment” or “segment” is a portion of an amino acid sequence,comprising at least one amino acid, or a portion of a nucleic acidsequence comprising at least one nucleotide. The terms “fragment” and“segment” are used interchangeably herein.

As used herein, the term “fragment,” as applied to a protein or peptide,can ordinarily be at least about 2-15 amino acids in length, at leastabout 15-25 amino acids, at least about 25-50 amino acids in length, atleast about 50-75 amino acids in length, at least about 75-100 aminoacids in length, and greater than 100 amino acids in length, dependingon the particular protein or peptide being referred to.

As used herein, the term “fragment” as applied to a nucleic acid, mayordinarily be at least about 20 nucleotides in length, typically, atleast about 50 nucleotides, more typically, from about 50 to about 100nucleotides, preferably, at least about 100 to about 200 nucleotides,even more preferably, at least about 200 nucleotides to about 300nucleotides, yet even more preferably, at least about 300 to about 350,even more preferably, at least about 350 nucleotides to about 500nucleotides, yet even more preferably, at least about 500 to about 600,even more preferably, at least about 600 nucleotides to about 620nucleotides, yet even more preferably, at least about 620 to about 650,and most preferably, the nucleic acid fragment will be greater thanabout 650 nucleotides in length.

As used herein, a “functional” molecule is a molecule in a form in whichit exhibits a property or activity by which it is characterized. Afunctional enzyme, for example, is one that exhibits the characteristiccatalytic activity by which the enzyme is characterized.

“Homologous” as used herein, refers to the subunit sequence similaritybetween two polymeric molecules, e.g., between two nucleic acidmolecules, e.g., two DNA molecules or two RNA molecules, or between twopolypeptide molecules. When a subunit position in both of the twomolecules is occupied by the same monomeric subunit, e.g., if a positionin each of two DNA molecules is occupied by adenine, then they arehomologous at that position. The homology between two sequences is adirect function of the number of matching or homologous positions, e.g.,if half (e.g., five positions in a polymer ten subunits in length) ofthe positions in two compound sequences are homologous then the twosequences are 50% homologous, if 90% of the positions, e.g., 9 of 10,are matched or homologous, the two sequences share 90% homology. By wayof example, the DNA sequences 3ATTGCCS' and 3′ TATGGC share 50%homology.

As used herein, “homology” is used synonymously with “identity.”

The determination of percent identity between two nucleotide or aminoacid sequences can be accomplished using a mathematical algorithm. Forexample, a mathematical algorithm useful for comparing two sequences isthe algorithm of Karlin and Altschul (1990, Proc. Natl. Acad. Sci. USA87:2264-2268), modified as in Karlin and Altschul (1993, Proc. Natl.Acad. Sci. USA 90:5873-5877). This algorithm is incorporated into theNBLAST and)(BLAST programs of Altschul, et al. (1990, J. Mol. Biol.215:403-410), and can be accessed, for example at the National Centerfor Biotechnology Information (NCBI) world wide web site. BLASTnucleotide searches can be performed with the NBLAST program (designated“blastn” at the NCBI web site), using the following parameters: gappenalty=5; gap extension penalty=2; mismatch penalty=3; match reward=1;expectation value 10.0; and word size=1 1 to obtain nucleotide sequenceshomologous to a nucleic acid described herein. BLAST protein searchescan be performed with the)(BLAST program (designated “blastn” at theNCBI web site) or the NCBI “blastp” program, using the followingparameters: expectation value 10.0, BLOSUM62 scoring matrix to obtainamino acid sequences homologous to a protein molecule described herein.To obtain gapped alignments for comparison purposes, Gapped BLAST can beutilized as described in Altschul et al. (1997, Nucleic Acids Res.25:3389-3402). Alternatively, PSI-Blast or PHI-Blast can be used toperform an iterated search which detects distant relationships betweenmolecules (Id.) and relationships between molecules which share a commonpattern. When utilizing BLAST, Gapped BLAST, PSI-Blast, and PHI-Blastprograms, the default parameters of the respective programs (e.g.,XBLAST and NBLAST) can be used.

The percent identity between two sequences can be determined usingtechniques similar to those described above, with or without allowinggaps. In calculating percent identity, typically exact matches arecounted.

As used herein, the term “hybridization” is used in reference to thepairing of complementary nucleic acids. Hybridization and the strengthof hybridization (i.e., the strength of the association between thenucleic acids) is impacted by such factors as the degree ofcomplementarity between the nucleic acids, stringency of the conditionsinvolved, the length of the formed hybrid, and the G:C ratio within thenucleic acids.

As used herein, the term “induction of apoptosis” means a process bywhich a cell is affected in such a way that it begins the process ofprogrammed cell death, which is characterized by the fragmentation ofthe cell into membrane-bound particles that are subsequently eliminatedby the process of phagocytosis.

The term “inhibit,” as used herein, refers to the ability of a compound,agent, or method to reduce or impede a described function, level,activity, rate, etc., based on the context in which the term “inhibit”is used. Preferably, inhibition is by at least 10%, more preferably byat least 25%, even more preferably by at least 50%, and most preferably,the function is inhibited by at least 75%. The term “inhibit” is usedinterchangeably with “reduce” and “block.”

The term “inhibit a protein,” as used herein, refers to any method ortechnique which inhibits protein synthesis, levels, activity, orfunction, as well as methods of inhibiting the induction or stimulationof synthesis, levels, activity, or function of the protein of interest.The term also refers to any metabolic or regulatory pathway which canregulate the synthesis, levels, activity, or function of the protein ofinterest. The term includes binding with other molecules and complexformation. Therefore, the term “protein inhibitor” refers to any agentor compound, the application of which results in the inhibition ofprotein function or protein pathway function. However, the term does notimply that each and every one of these functions must be inhibited atthe same time.

As used herein “injecting or applying” includes administration of acompound of the invention by any number of routes and means including,but not limited to, topical, oral, buccal, intravenous, intramuscular,intra arterial, intramedullary, intrathecal, intraventricular,transdermal, subcutaneous, intraperitoneal, intranasal, enteral,topical, sublingual, vaginal, ophthalmic, pulmonary, or rectal means.

The term “injected once with a 5-daily dose”, as used herein, means thatan induction therapy was initiated wherein mice were injected with 1 μgprotein once a day for five consecutive days and then followed over timeas indicated.

As used herein, an “instructional material” includes a publication, arecording, a diagram, or any other medium of expression which can beused to communicate the usefulness of the peptide of the invention inthe kit for effecting alleviation of the various diseases or disordersrecited herein. Optionally, or alternately, the instructional materialmay describe one or more methods of alleviating the diseases ordisorders in a cell or a tissue of a mammal. The instructional materialof the kit of the invention may, for example, be affixed to a containerwhich contains the identified compound invention or be shipped togetherwith a container which contains the identified compound. Alternatively,the instructional material may be shipped separately from the containerwith the intention that the instructional material and the compound beused cooperatively by the recipient.

The term “ischemia” as used herein refers to a local anemia due tomechanical obstruction of the blood supply, which gives rise toinadequate circulation of the blood to an organ, tissue, or region of anorgan or tissue.

An “isolated nucleic acid” refers to a nucleic acid segment or fragmentwhich has been separated from sequences which flank it in a naturallyoccurring state, e.g., a DNA fragment which has been removed from thesequences which are normally adjacent to the fragment, e.g., thesequences adjacent to the fragment in a genome in which it naturallyoccurs. The term also applies to nucleic acids which have beensubstantially purified from other components which naturally accompanythe nucleic acid, e.g., RNA or DNA or proteins, which naturallyaccompany it in the cell. The term therefore includes, for example, arecombinant DNA which is incorporated into a vector, into anautonomously replicating plasmid or virus, or into the genomic DNA of aprokaryote or eukaryote, or which exists as a separate molecule (e.g.,as a cDNA or a genomic or cDNA fragment produced by PCR or restrictionenzyme digestion) independent of other sequences. It also includes arecombinant DNA which is part of a hybrid gene encoding additionalpolypeptide sequence.

A “ligand” is a compound that specifically binds to a target receptor.

A “receptor” is a compound that specifically binds to a ligand.

As used herein, the term “linkage” refers to a connection between twogroups. The connection can be either covalent or non-covalent, includingbut not limited to ionic bonds, hydrogen bonding, andhydrophobic/hydrophilic interactions.

As used herein, the term “linker” refers to a molecule that joins twoother molecules either covalently or noncovalently, e.g., through ionicor hydrogen bonds or van der Waals interactions.

“Malexpression” of a gene means expression of a gene in a cell of apatient afflicted with a disease or disorder, wherein the level ofexpression (including non-expression), the portion of the geneexpressed, or the timing of the expression of the gene with regard tothe cell cycle, differs from expression of the same gene in a cell of apatient not afflicted with the disease or disorder. It is understoodthat malexpression may cause or contribute to the disease or disorder,be a symptom of the disease or disorder, or both.

The term “material” refers to any compound, molecule, substance, orgroup or combination thereof that forms any type of structure or groupof structures during or after electroprocessing. Materials includenatural materials, synthetic materials, or combinations thereof.Naturally occurring organic materials include any substances naturallyfound in the body of plants or other organisms, regardless of whetherthose materials have or can be produced or altered synthetically.Synthetic materials include any materials prepared through any method ofartificial synthesis, processing, or manufacture. Preferably, thematerials are biologically compatible materials.

The term “measuring the level of expression” or “determining the levelof expression” as used herein refers to any measure or assay which canbe used to correlate the results of the assay with the level ofexpression of a gene or protein of interest. Such assays includemeasuring the level of mRNA, protein levels, etc., and can be performedby assays such as northern and western blot analyses, binding assays,immunoblots, etc. The level of expression can include rates ofexpression and can be measured in terms of the actual amount of an mRNAor protein present. Such assays are coupled with processes or systems tostore and process information and to help quantify levels, signals, etc.and to digitize the information for use in comparing levels.

The term “modulate”, as used herein, refers to changing the level of anactivity, function, or process. The term “modulate” encompasses bothinhibiting and stimulating an activity, function, or process.

Unless otherwise specified, a “nucleotide sequence encoding an aminoacid sequence” includes all nucleotide sequences that are degenerateversions of each other and that encode the same amino acid sequence.Nucleotide sequences that encode proteins and RNA may include introns.

As used herein, the term “nucleic acid” encompasses RNA as well assingle and double-stranded DNA and cDNA. Furthermore, the terms,“nucleic acid,” “DNA,” “RNA” and similar terms also include nucleic acidanalogs, i.e. analogs having other than a phosphodiester backbone. Forexample, the so-called “peptide nucleic acids,” which are known in theart and have peptide bonds instead of phosphodiester bonds in thebackbone, are considered within the scope of the present invention. By“nucleic acid” is meant any nucleic acid, whether composed ofdeoxyribonucleosides or ribonucleosides, and whether composed ofphosphodiester linkages or modified linkages such as phosphotriester,phosphoramidate, siloxane, carbonate, carboxymethylester, acetamidate,carbamate, thioether, bridged phosphoramidate, bridged methylenephosphonate, bridged phosphoramidate, bridged phosphoramidate, bridgedmethylene phosphonate, phosphorothioate, methylphosphonate,phosphorodithioate, bridged phosphorothioate or sulfone linkages, andcombinations of such linkages. The term nucleic acid also specificallyincludes nucleic acids composed of bases other than the fivebiologically occurring bases (adenine, guanine, thymine, cytosine anduracil). Conventional notation is used herein to describe polynucleotidesequences: the left-hand end of a single-stranded polynucleotidesequence is the 5′-end; the left-hand direction of a double-strandedpolynucleotide sequence is referred to as the 5′-direction. Thedirection of 5′ to 3′ addition of nucleotides to nascent RNA transcriptsis referred to as the transcription direction. The DNA strand having thesame sequence as an mRNA is referred to as the “coding strand”;sequences on the DNA strand which are located 5′ to a reference point onthe DNA are referred to as “upstream sequences”; sequences on the DNAstrand which are 3′ to a reference point on the DNA are referred to as“downstream sequences.”

The term “nucleic acid construct,” as used herein, encompasses DNA andRNA sequences encoding the particular gene or gene fragment desired,whether obtained by genomic or synthetic methods.

Unless otherwise specified, a “nucleotide sequence encoding an aminoacid sequence” includes all nucleotide sequences that are degenerateversions of each other and that encode the same amino acid sequence.Nucleotide sequences that encode proteins and RNA may include introns.

The term “Oligonucleotide” typically refers to short polynucleotides,generally no greater than about 50 nucleotides. It will be understoodthat when a nucleotide sequence is represented by a DNA sequence (i.e.,A, T, G, C), this also includes an RNA sequence (i.e., A, U, G, C) inwhich “U” replaces “T.” “Operably linked” refers to a juxtapositionwherein the components are configured so as to perform their usualfunction. Thus, control sequences or promoters operably linked to acoding sequence are capable of effecting the expression of the codingsequence. By describing two polynucleotides as “operably linked” ismeant that a single-stranded or double-stranded nucleic acid moietycomprises the two polynucleotides arranged within the nucleic acidmoiety in such a manner that at least one of the two polynucleotides isable to exert a physiological effect by which it is characterized uponthe other. By way of example, a promoter operably linked to the codingregion of a gene is able to promote transcription of the coding region.

As used herein, “parenteral administration” of a pharmaceuticalcomposition includes any route of administration characterized byphysical breaching of a tissue of a subject and administration of thepharmaceutical composition through the breach in the tissue. Parenteraladministration thus includes, but is not limited to, administration of apharmaceutical composition by injection of the composition, byapplication of the composition through a surgical incision, byapplication of the composition through a tissue-penetrating non-surgicalwound, and the like. In particular, parenteral administration iscontemplated to include, but is not limited to, subcutaneous,intraperitoneal, intramuscular, intrasternal injection, and kidneydialytic infusion techniques.

The term “peptide” typically refers to short polypeptides.

The term “per application” as used herein refers to administration of acompositions, drug, or compound to a subject.

“Permeation enhancement” and “permeation enhancers” as used hereinrelate to the process and added materials which bring about an increasein the permeability of skin to a poorly skin permeatingpharmacologically active agent, i.e., so as to increase the rate atwhich the drug permeates through the skin and enters the bloodstream.“Permeation enhancer” is used interchangeably with “penetrationenhancer”.

The term “pharmaceutical composition” shall mean a compositioncomprising at least one active ingredient, whereby the composition isamenable to investigation for a specified, efficacious outcome in amammal (for example, without limitation, a human). Those of ordinaryskill in the art will understand and appreciate the techniquesappropriate for determining whether an active ingredient has a desiredefficacious outcome based upon the needs of the artisan. As used herein,“pharmaceutical compositions” include formulations for human andveterinary use.

As used herein, the term “pharmaceutically-acceptable carrier” means achemical composition with which an appropriate compound or derivativecan be combined and which, following the combination, can be used toadminister the appropriate compound to a subject.

As used herein, the term “physiologically acceptable” ester or saltmeans an ester or salt form of the active ingredient which is compatiblewith any other ingredients of the pharmaceutical composition, which isnot deleterious to the subject to which the composition is to beadministered.

“Plurality” means at least two.

A “polynucleotide” means a single strand or parallel and anti-parallelstrands of a nucleic acid. Thus, a polynucleotide may be either asingle-stranded or a double-stranded nucleic acid.

“Polypeptide” refers to a polymer composed of amino acid residues,related naturally occurring structural variants, and syntheticnon-naturally occurring analogs thereof linked via peptide bonds,related naturally occurring structural variants, and syntheticnon-naturally occurring analogs thereof.

“Synthetic peptides or polypeptides” means a non-naturally occurringpeptide or polypeptide. Synthetic peptides or polypeptides can besynthesized, for example, using an automated polypeptide synthesizer.Various solid phase peptide synthesis methods are known to those ofskill in the art.

By “presensitization” is meant pre-administration of at least one innateimmune system stimulator prior to challenge with a pathogenic agent.This is sometimes referred to as induction of tolerance.

The term “prevent,” as used herein, means to stop something fromhappening, or taking advance measures against something possible orprobable from happening. In the context of medicine, “prevention”generally refers to action taken to decrease the chance of getting adisease or condition.

A “preventive” or “prophylactic” treatment is a treatment administeredto a subject who does not exhibit signs, or exhibits only early signs,of a disease or disorder. A prophylactic or preventative treatment isadministered for the purpose of decreasing the risk of developingpathology associated with developing the disease or disorder.

“Primer” refers to a polynucleotide that is capable of specificallyhybridizing to a designated polynucleotide template and providing apoint of initiation for synthesis of a complementary polynucleotide.Such synthesis occurs when the polynucleotide primer is placed underconditions in which synthesis is induced, i.e., in the presence ofnucleotides, a complementary polynucleotide template, and an agent forpolymerization such as DNA polymerase. A primer is typicallysingle-stranded, but may be double-stranded. Primers are typicallydeoxyribonucleic acids, but a wide variety of synthetic and naturallyoccurring primers are useful for many applications. A primer iscomplementary to the template to which it is designed to hybridize toserve as a site for the initiation of synthesis, but need not reflectthe exact sequence of the template. In such a case, specifichybridization of the primer to the template depends on the stringency ofthe hybridization conditions. Primers can be labeled with, e.g.,chromogenic, radioactive, or fluorescent moieties and used as detectablemoieties.

As used herein, the term “promoter/regulatory sequence” means a nucleicacid sequence which is required for expression of a gene productoperably linked to the promo ter/regulator sequence. In some instances,this sequence may be the core promoter sequence and in other instances,this sequence may also include an enhancer sequence and other regulatoryelements which are required for expression of the gene product. Thepromoter/regulatory sequence may, for example, be one which expressesthe gene product in a tissue specific manner.

A “constitutive” promoter is a promoter which drives expression of agene to which it is operably linked, in a constant manner in a cell. Byway of example, promoters which drive expression of cellularhousekeeping genes are considered to be constitutive promoters.

An “inducible” promoter is a nucleotide sequence which, when operablylinked with a polynucleotide which encodes or specifies a gene product,causes the gene product to be produced in a living cell substantiallyonly when an inducer which corresponds to the promoter is present in thecell.

A “tissue-specific” promoter is a nucleotide sequence which, whenoperably linked with a polynucleotide which encodes or specifies a geneproduct, causes the gene product to be produced in a living cellsubstantially only if the cell is a cell of the tissue typecorresponding to the promoter.

As used herein, “protecting group” with respect to a terminal aminogroup refers to a terminal amino group of a peptide, which terminalamino group is coupled with any of various amino-terminal protectinggroups traditionally employed in peptide synthesis. Such protectinggroups include, for example, acyl protecting groups such as formyl,acetyl, benzoyl, trifluoroacetyl, succinyl, and methoxysuccinyl;aromatic urethane protecting groups such as benzyloxycarbonyl; andaliphatic urethane protecting groups, for example, tert-butoxycarbonylor adamantyloxycarbonyl. See Gross and Mienhofer, eds., The Peptides,vol. 3, pp. 3-88 (Academic Press, New York, 1981) for suitableprotecting groups.

As used herein, “protecting group” with respect to a terminal carboxygroup refers to a terminal carboxyl group of a peptide, which terminalcarboxyl group is coupled with any of various carboxyl-terminalprotecting groups. Such protecting groups include, for example,tert-butyl, benzyl or other acceptable groups linked to the terminalcarboxyl group through an ester or ether bond.

The term “prevent,” as used herein, means to stop something fromhappening, or taking advance measures against something possible orprobable from happening. In the context of medicine, “prevention”generally refers to action taken to decrease the chance of getting adisease or condition.

A “prophylactic” treatment is a treatment administered to a subject whodoes not exhibit signs of a disease or injury or exhibits only earlysigns of the disease or injury for the purpose of decreasing the risk ofdeveloping pathology associated with the disease or injury.

As used herein, the term “purified” and like terms relate to anenrichment of a molecule or compound relative to other componentsnormally associated with the molecule or compound in a nativeenvironment. The term “purified” does not necessarily indicate thatcomplete purity of the particular molecule has been achieved during theprocess. A “highly purified” compound as used herein refers to acompound that is greater than 90% pure. In particular, purified spermcell DNA refers to DNA that does not produce significant detectablelevels of non-sperm cell DNA upon PCR amplification of the purifiedsperm cell DNA and subsequent analysis of that amplified DNA. A“significant detectable level” is an amount of contaminate that would bevisible in the presented data and would need to be addressed/explainedduring analysis of the forensic evidence.

The term “protein regulatory pathway”, as used herein, refers to boththe upstream regulatory pathway which regulates a protein, as well asthe downstream events which that protein regulates. Such regulationincludes, but is not limited to, transcription, translation, levels,activity, posttranslational modification, and function of the protein ofinterest, as well as the downstream events which the protein regulates.The terms “protein pathway” and “protein regulatory pathway” are usedinterchangeably herein.

“Recombinant polynucleotide” refers to a polynucleotide having sequencesthat are not naturally joined together. An amplified or assembledrecombinant polynucleotide may be included in a suitable vector, and thevector can be used to transform a suitable host cell.

A recombinant polynucleotide may serve a non-coding function (e.g.,promoter, origin of replication, ribosome-binding site, etc.) as well.

A host cell that comprises a recombinant polynucleotide is referred toas a “recombinant host cell.” A gene which is expressed in a recombinanthost cell wherein the gene comprises a recombinant polynucleotide,produces a “recombinant polypeptide.”

A “recombinant polypeptide” is one which is produced upon expression ofa recombinant polynucleotide.

The term “regulate” refers to either stimulating or inhibiting afunction or activity of interest.

A “sample,” as used herein, refers preferably to a biological samplefrom a subject, including, but not limited to, normal tissue samples,diseased tissue samples, biopsies, blood, saliva, feces, semen, tears,and urine. A sample can also be any other source of material obtainedfrom a subject which contains cells, tissues, or fluid of interest. Asample can also be obtained from cell or tissue culture.

As used herein, the term “secondary antibody” refers to an antibody thatbinds to the constant region of another antibody (the primary antibody).

By the term “signal sequence” is meant a polynucleotide sequence whichencodes a peptide that directs the path a polypeptide takes within acell, i.e., it directs the cellular processing of a polypeptide in acell, including, but not limited to, eventual secretion of a polypeptidefrom a cell. A signal sequence is a sequence of amino acids which aretypically, but not exclusively, found at the amino terminus of apolypeptide which targets the synthesis of the polypeptide to theendoplasmic reticulum. In some instances, the signal peptide isproteolytically removed from the polypeptide and is thus absent from themature protein.

By “small interfering RNAs (siRNAs)” is meant, inter alia, an isolateddsRNA molecule comprised of both a sense and an anti-sense strand. Inone aspect, it is greater than 10 nucleotides in length. siRNA alsorefers to a single transcript which has both the sense and complementaryantisense sequences from the target gene, e.g., a hairpin. siRNA furtherincludes any form of dsRNA (proteolytically cleaved products of largerdsRNA, partially purified RNA, essentially pure RNA, synthetic R A,recombinantly produced RNA) as well as altered RNA that differs fromnaturally occurring RNA by the addition, deletion, substitution, and/oralteration of one or more nucleotides.

As used herein, the term “solid support” relates to a solvent insolublesubstrate that is capable of forming linkages (preferably covalentbonds) with various compounds. The support can be either biological innature, such as, without limitation, a cell or bacteriophage particle,or synthetic, such as, without limitation, an acrylamide derivative,agarose, cellulose, nylon, silica, or magnetized particles.

By the term “specifically binds to”, as used herein, is meant when acompound or ligand functions in a binding reaction or assay conditionswhich is determinative of the presence of the compound in a sample ofheterogeneous compounds.

The term “standard,” as used herein, refers to something used forcomparison. For example, a standard can be a known standard agent orcompound which is administered or added to a control sample and used forcomparing results when measuring said compound in a test sample. In oneaspect, the standard compound is added or prepared at an amount orconcentration that is equivalent to a normal value for that compound ina normal subject. Standard can also refer to an “internal standard,”such as an agent or compound which is added at known amounts to a sampleand is useful in determining such things as purification or recoveryrates when a sample is processed or subjected to purification orextraction procedures before a marker of interest is measured. Internalstandards are often a purified marker of interest which has beenlabeled, such as with a radioactive isotope, allowing it to bedistinguished from an endogenous marker.

A “subject” of analysis, diagnosis, or treatment is an animal. Suchanimals include mammals, preferably a human.

As used herein, a “subject in need thereof” is a patient, animal,mammal, or human, who will benefit from the method of this invention.

As used herein, a “substantially homologous amino acid sequence”includes those amino acid sequences which have at least about 95%homology, preferably at least about 96% homology, more preferably atleast about 97% homology, even more preferably at least about 98%homology, and most preferably at least about 99% homology to an aminoacid sequence of a reference sequence. Amino acid sequences similarityor identity can be computed using, for example, the BLASTP and TBLASTNprograms which employ the BLAST (basic local alignment search tool)algorithm. The default setting used for these programs are suitable foridentifying substantially similar amino acid sequences for purposes ofthe present invention.

“Substantially homologous nucleic acid sequence” means a nucleic acidsequence corresponding to a reference nucleic acid sequence wherein thecorresponding sequence encodes a peptide having substantially the samestructure and function as the peptide encoded by the reference nucleicacid sequence; e.g., where only changes in amino acids not significantlyaffecting the peptide function occur. Preferably, the substantiallysimilar nucleic acid sequence encodes the peptide encoded by thereference nucleic acid sequence. The percentage of identity between thesubstantially similar nucleic acid sequence and the reference nucleicacid sequence is at least about 50%, 65%, 75%, 85%, 95%, 96%, 97%, 98%,99% or more. Substantial similarity of nucleic acid sequences can bedetermined by comparing the sequence identity of two sequences, forexample by physical chemical methods (i.e., hybridization) or bysequence alignment via computer algorithm. Suitable nucleic acidhybridization conditions to determine if a nucleotide sequence issubstantially similar to a reference nucleotide sequence are: 7% sodiumdodecyl sulfate SDS, 0.5 M NaPC>4, 1 mM EDTA at 50° C. with washing in2×standard saline citrate (SSC), 0.1% SDS at 50° C.; preferably in 7%(SDS), 0.5 M NaPO4, 1 mM EDTA at 50° C. with washing in 1×SSC, 0.1% SDSat 50° C.; preferably 7% SDS, 0.5 M NaPO4, 1 mM EDTA at 50° C. withwashing in 0.5×SSC, 0.1% SDS at 50° C.; and more preferably in 7% SDS,0.5 M NaPO4, 1 mM EDTA at 50° C. with washing in 0.1×SSC, 0.1% SDS at65° C. Suitable computer algorithms to determine substantial similaritybetween two nucleic acid sequences include, GCS program package(Devereux et al., 1984 Nucl. Acids Res. 12:387), and the BLASTN or FASTAprograms (Altschul et al, 1990 Proc. Natl. Acad. Sci. USA. 1990 87:14:5509-13; Altschul et al, J. Mol. Biol. 1990 215:3:403-10; Altschul etal, 1997 Nucleic Acids Res. 25:3389-3402). The default settings providedwith these programs are suitable for determining substantial similarityof nucleic acid sequences for purposes of the present invention.

The term “substantially pure” describes a compound, e.g., a protein orpolypeptide which has been separated from components which naturallyaccompany it. Typically, a compound is substantially pure when at least10%, more preferably at least 20%, more preferably at least 50%, morepreferably at least 60%, more preferably at least 75%, more preferablyat least 90%, and most preferably at least 99% of the total material (byvolume, by wet or dry weight, or by mole percent or mole fraction) in asample is the compound of interest. Purity can be measured by anyappropriate method, e.g., in the case of polypeptides by columnchromatography, gel electrophoresis, or HPLC analysis. A compound, e.g.,a protein, is also substantially purified when it is essentially free ofnaturally associated components or when it is separated from the nativecontaminants which accompany it in its natural state.

The term “symptom,” as used herein, refers to any morbid phenomenon ordeparture from the normal in structure, function, or sensation,experienced by the patient and indicative of disease. In contrast, a“sign” is objective evidence of disease. For example, a bloody nose is asign. It is evident to the patient, doctor, nurse and other observers.

A “therapeutic” treatment is a treatment administered to a subject whoexhibits signs of pathology for the purpose of diminishing oreliminating those signs.

A “therapeutically effective amount” of a compound is that amount ofcompound which is sufficient to provide a beneficial effect to thesubject to which the compound is administered.

“Tissue” means (1) a group of similar cells united to perform a specificfunction; (2) a part of an organism consisting of an aggregate of cellshaving a similar structure and function; or (3) a grouping of cells thatare similarly characterized by their structure and function, such asmuscle or nerve tissue.

The term “transfection” is used interchangeably with the terms “genetransfer”, “transformation,” and “transduction”, and means theintracellular introduction of a polynucleotide. “Transfectionefficiency” refers to the relative amount of the transgene taken up bythe cells subjected to transfection. In practice, transfectionefficiency is estimated by the amount of the reporter gene productexpressed following the transfection procedure.

The term “transgene” is used interchangeably with “inserted gene,” or“expressed gene” and, where appropriate, “gene”. “Transgene” refers to apolynucleotide that, when introduced into a cell, is capable of beingtranscribed under appropriate conditions so as to confer a beneficialproperty to the cell such as, for example, expression of atherapeutically useful protein. It is an exogenous nucleic acid sequencecomprising a nucleic acid which encodes a promoter/regulatory sequenceoperably linked to nucleic acid which encodes an amino acid sequence,which exogenous nucleic acid is encoded by a transgenic mammal.

As used herein, a “transgenic cell” is any cell that comprises a nucleicacid sequence that has been introduced into the cell in a manner thatallows expression of a gene encoded by the introduced nucleic acidsequence.

As used herein, the term “transgenic mammal” means a mammal, the germcells of which comprise an exogenous nucleic acid.

The term to “treat,” as used herein, means reducing the frequency withwhich symptoms are experienced by a patient or subject or administeringan agent or compound to reduce the frequency with which symptoms areexperienced.

As used herein, the term “treating” may include prophylaxis of thespecific injury, disease, disorder, or condition, or alleviation of thesymptoms associated with a specific injury, disease, disorder, orcondition and/or preventing or eliminating said symptoms. A“prophylactic” treatment is a treatment administered to a subject whodoes not exhibit signs of a disease or exhibits only early signs of thedisease for the purpose of decreasing the risk of developing pathologyassociated with the disease and should be interpreted based on thecontext of the use.

“Treating” is used interchangeably with “treatment” herein.

A “vector” is a composition of matter which comprises an isolatednucleic acid and which can be used to deliver the isolated nucleic acidto the interior of a cell. Numerous vectors are known in the artincluding, but not limited to, linear polynucleotides, polynucleotidesassociated with ionic or amphiphilic compounds, plasmids, and viruses.Thus, the term “vector” includes an autonomously replicating plasmid ora virus. The term should also be construed to include non-plasmid andnon-viral compounds which facilitate transfer or delivery of nucleicacid to cells, such as, for example, polylysine compounds, liposomes,and the like. Examples of viral vectors include, but are not limited to,adenoviral vectors, adeno-associated virus vectors, retroviral vectors,recombinant viral vectors, and the like. Examples of non-viral vectorsinclude, but are not limited to, liposomes, polyamine derivatives of DNAand the like.

“Expression vector” refers to a vector comprising a recombinantpolynucleotide comprising expression control sequences operativelylinked to a nucleotide sequence to be expressed. An expression vectorcomprises sufficient cis-acting elements for expression; other elementsfor expression can be supplied by the host cell or in an in vitroexpression system. Expression vectors include all those known in theart, such as cosmids, plasmids (e.g., naked or contained in liposomes)and viruses that incorporate the recombinant polynucleotide.

Embodiments

The present application discloses the unexpected result of synergybetween IL-2 and IL-33 as a combination therapy and an even betterresult/efficacy using a novel IL-2/IL-33 fusion protein. In someaspects, the results can be additive. In some aspects, the results aresynergistic.

In one embodiment, the IL233 fusion protein can be used for treatment ofinflammatory conditions via boosting the homeostasis and activity ofTreg cells, which offer the major mechanism of peripheral immunetolerance.

In another embodiment, the IL233 fusion protein can be used topotentiate the Th2 response by increasing the production of the relatedcytokines (IL-4, IL-5, and IL-13) by the differentiated Th2 cells, theinnate lymphoid cells, Nuocytes, natural helper cells or fat associatedlymphoid cluster (FALC).

In yet another embodiment, the invention can be used for skewing of theimmune response by inhibiting the pro-inflammatory Th1 response andboosting the anti-inflammatory Th2 immune response.

In a further embodiment, the invention can be used for altering thephenotype of innate immune cells, such as dendritic cells (DC) andmacrophages to an altered maturation phenotype, such that these innateimmune cells can skew the differentiation of immune response toward aTh2 response, while suppressing the Th1 differentiation. The IL233treated DC and macrophages can also be employed for boosting thehomeostasis of Treg cells.

In one embodiment, the compositions and methods of the invention areuseful for regulating the above described mechanisms either individuallyor in cooperation for prevention or treatment of autoimmune disordersincluding Type-1 diabetes, Type-2 diabetes, multiple sclerosis,atherosclerosis, systemic lupus erythematosus (lupus), autoimmunepancreatitis, IgG4-related systemic disease spectrum diseases(IgG4-RSD), Sjogren's syndrome, inflammatory bowel disease (Crohn'sdisease, and ulcerative colitis), autoimmune thyroiditis, autoimmuneencephalomyelitis, Alzheimer's disease and dementia, ankylosingspondylitis, chronic obstructive pulmonary disease, myasthenia gravis,obesity, osteoporosis, periodontal disease, psoriasis and uveitis.

In another embodiment, the IL233 fusion protein can be administered toboost immune tolerance during transplantation and to enhancing graftsurvival via boosting the homeostasis and recruitment of Treg and Th2cells. In yet another embodiment, the compositions and methods of theinvention can be employed for treatment of inflammatory conditionsarising due to ischemia reperfusion injury of various organs including,kidneys, lung and heart.

In a further embodiment, the compositions and methods of the inventionare useful for treating cardiovascular diseases by not only suppressinginflammation, but also by improving cardiovascular function. Treatmentwith IL-33 has been demonstrated to have cardio-protective properties ina model of pressure overload and improved survival following transverseaortic constriction in wild-type but not IL-33 receptor mice. IL-33 canalso reduce cardiomyocyte apoptosis, decrease infarct and fibrosis, andimprove ventricular function in vivo via suppression of caspase-3activity and increased expression of the ‘inhibitor of apoptosis’ familyof proteins.

In one embodiment, the compositions and methods of the invention areuseful for suppressing inflammation in the central nervous system andthey are useful for promoting the homeostasis of microglia cells. Themethods include treatment of several neurological disorders linked withmicroglia-cell related defects such as Rhett syndrome.

In one embodiment, the compositions and methods of the invention areuseful for reducing inflammation in adipose tissue by altering thephenotype of the adipose tissue resident macrophages to an alteredmaturation phenotype, which will suppress the Th1 and Th17 inducedinflammation associated with obesity and Type-2 diabetes. The fusionprotein or combinations of 11-2 and IL-33 will also be useful forsuppression of adipogenesis, because IL-33 has been shown to suppressthe expression of several genes related to adipogenesis and improvefasting glucose levels as well as resistance to insulin and glucose inmouse model of type-2 diabetes.

One of ordinary skill in the art will appreciate that the sequences ofthe components of the IL233 fusion protein can be modified independentlyof one another with conservative amino acid changes, including,insertions, deletions, and substitutions, and that the valency could bealtered as well, as long as the resulting multimer/multimeric complexremains effective. Amino acid changes (fragments and homologs) can bemade independently in each IL-2 and IL-33 as well when they are beingused in combination therapy.

In one aspect, a fusion protein construct of the invention or a IL-2 andIL-33 combination can be administered by a route selected from,including, but not limited to, intravenously, intrathecally, locally,intramuscularly, topically, orally, intra-arterially, parenterally, etc.Administration can be more than once. One of ordinary skill in the artcan determine how often to administer the compound, the dose to be used,and what combination of other agents it can be administered with such astherapeutic agents and/or other drugs or compounds such as antimicrobialagents, anti-inflammatory agents, etc. One of ordinary skill in the artcan also determine if all compounds should be administeredsimultaneously or not.

In one embodiment, a peptide construct dosage or protein dosage of about0.1 μg/kg body weight to about 100 mg/kg can be administered to asubject in need thereof, including whole numbers between 0.1 and 100 andfractions thereof. In one aspect, a peptide construct dosage or proteindosage of about 0.1 μg/kg to about 75 μg/kg can be administered to asubject. In another aspect, a dosage of about 5.0 μg/kg to about 50μg/kg can be administered to a subject. In yet another aspect, a dosageof about 10 μg/kg to about 25 μg/kg can be administered to a subject. Ina further aspect, a dosage of about 15 μg/kg to about 20 μg/kg can beadministered to a subject. In one aspect, a dose of about 1 μg/kg bodyweight to about 1000 μg/kg body weight, or about, 10 μkg body weight toabout 500 μg/kg body weight, about 20 μg/kg body weight to about 100μg/kg body weight, or about 30 μg/kg body weight to about 50 μg/kg bodyweight. In one aspect, doses that can be used include 5.0, 15, 50, and150 μg/kg of body weight.

Numerical ranges recited herein by endpoints include all numbers andfractions subsumed within that range (e.g., 1 to 5 includes 1, 1.5, 2,2.75, 3, 3.90, 4, and 5). It is also to be understood that all numbersand fractions thereof are presumed to be modified by the term “about.”

In one embodiment, a unit dose of fusion protein construct or proteinscan be administered. Other therapeutic agents of the invention can alsobe administered as unit doses. Kits can be provided with unit doses in acontainer or syringe or amounts that one of ordinary skill in the artcan administer based on a dose per weight, etc.

In one embodiment, a fusion protein construct or proteins of theinvention are administered at least once a day, or at least once a week,or at least once a month. In one embodiment, a fusion peptide constructor proteins of the invention are administered at least twice a day, atleast twice a week, or at least twice a month. In one aspect, doses areadministered in a series of five doses over five days. One of ordinaryskill in the art can determine how much to administer and how often toadminister it.

The invention further includes isolated nucleic acids comprisingsequences encoding proteins or peptides of the invention. The presentinvention further includes a fusion protein wherein the order of thepeptides is reversed. In one aspect, the IL-33 is fused to theN-terminus of IL-2 with or without a linker sequence. In another aspect,the IL-2 is fused to the N-terminus of IL-33 with or without a linkersequence. One of ordinary skill in the art will appreciate that thelinker length and sequence can be modified and that methods are providedfor easily test the resulting activity of the fusion peptide.

Also included are peptides and polypeptides which have been modifiedusing ordinary molecular biological techniques so as to improve theirresistance to proteolytic degradation or to optimize solubilityproperties or to render them more suitable as a therapeutic agent.Analogs of such polypeptides include those containing residues otherthan naturally occurring L-amino acids, e.g., D-amino acids ornon-naturally occurring or non-standard synthetic amino acids. Thepeptides of the invention are not limited to products of any of thespecific exemplary processes listed herein.

The invention includes the use of beta-alanine (also referred to asβ-alanine, β-Ala, bA and βA, having the structure:

Sequences are provided herein which use the symbol “PA”, but in theSequence Listing submitted herewith “βA” is provided as “Xaa” andreference in the text of the Sequence Listing indicates that Xaa is betaalanine.

The peptides of the present invention may be readily prepared bystandard, well-established techniques, such as solid-phase peptidesynthesis (SPPS) as described by Stewart et al. in Solid Phase PeptideSynthesis, 2nd Edition, 1984, Pierce Chemical Company, Rockford, Ill.;and as described by Bodanszky and Bodanszky in The Practice of PeptideSynthesis, 1984, Springer-Verlag, New York. At the outset, a suitablyprotected amino acid residue is attached through its carboxyl group to aderivatized, insoluble polymeric support, such as cross-linkedpolystyrene or polyamide resin. “Suitably protected” refers to thepresence of protecting groups on both the a-amino group of the aminoacid, and on any side chain functional groups. Side chain protectinggroups are generally stable to the solvents, reagents and reactionconditions used throughout the synthesis, and are removable underconditions which will not affect the final peptide product. Stepwisesynthesis of the oligopeptide is carried out by the removal of theN-protecting group from the initial amino acid, and couple thereto ofthe carboxyl end of the next amino acid in the sequence of the desiredpeptide. This amino acid is also suitably protected. The carboxyl of theincoming amino acid can be activated to react with the N-terminus of thesupport-bound amino acid by formation into a reactive group such asformation into a carbodiimide, a symmetric acid anhydride or an “activeester” group such as hydro xybenzotriazole or pentafluorophenly esters.

Examples of solid phase peptide synthesis methods include the BOC methodwhich utilized tert-butyloxcarbonyl as the a-amino protecting group, andthe FMOC method which utilizes 9-fluorenylmethyloxcarbonyl to protectthe a-amino of the amino acid residues, both methods of which are wellknown by those of skill in the art.

Incorporation of N- and/or C-blocking groups can also be achieved usingprotocols conventional to solid phase peptide synthesis methods. Forincorporation of C-terminal blocking groups, for example, synthesis ofthe desired peptide is typically performed using, as solid phase, asupporting resin that has been chemically modified so that cleavage fromthe resin results in a peptide having the desired C-terminal blockinggroup. To provide peptides in which the C-terminus bears a primary aminoblocking group, for instance, synthesis is performed using ap-methylbenzhydrylamine (MBHA) resin so that, when peptide synthesis iscompleted, treatment with hydrofluoric acid releases the desiredC-terminally amidated peptide. Similarly, incorporation of anN-methylamine blocking group at the C-terminus is achieved usingN-methylaminoethyl-derivatized DVB, resin, which upon HF treatmentreleases a peptide bearing an N-methylamidated C-terminus. Blockage ofthe C-terminus by esterification can also be achieved using conventionalprocedures. This entails use of resin/blocking group combination thatpermits release of side-chain peptide from the resin, to allow forsubsequent reaction with the desired alcohol, to form the esterfunction. FMOC protecting group, in combination with DVB resinderivatized with methoxyalkoxybenzyl alcohol or equivalent linker, canbe used for this purpose, with cleavage from the support being effectedby TFA in dichloromethane. Esterification of the suitably activatedcarboxyl function e.g. with DCC, can then proceed by addition of thedesired alcohol, followed by deprotection and isolation of theesterified peptide product.

Incorporation of N-terminal blocking groups can be achieved while thesynthesized peptide is still attached to the resin, for instance bytreatment with a suitable anhydride and nitrile. To incorporate anacetyl-blocking group at the N-terminus, for instance, the resin-coupledpeptide can be treated with 20% acetic anhydride in acetonitrile. TheN-blocked peptide product can then be cleaved from the resin,deprotected and subsequently isolated.

To ensure that the peptide obtained from either chemical or biologicalsynthetic techniques is the desired peptide, analysis of the peptidecomposition should be conducted. Such amino acid composition analysismay be conducted using high-resolution mass spectrometry to determinethe molecular weight of the peptide. Alternatively, or additionally, theamino acid content of the peptide can be confirmed by hydrolyzing thepeptide in aqueous acid, and separating, identifying and quantifying thecomponents of the mixture using HPLC, or an amino acid analyzer. Proteinsequenators, which sequentially degrade the peptide and identify theamino acids in order, may also be used to determine definitely thesequence of the peptide.

Prior to its use, the peptide is purified to remove contaminants. Inthis regard, it will be appreciated that the peptide will be purified soas to meet the standards set out by the appropriate regulatory agencies.Any one of a number of a conventional purification procedures may beused to attain the required level of purity including, for example,reversed-phase high-pressure liquid chromatography (HPLC) using analkylated silica column such as C4-, C8- or CI 8-silica. A gradientmobile phase of increasing organic content is generally used to achievepurification, for example, acetonitrile in an aqueous buffer, usuallycontaining a small amount of trifluoroacetic acid. Ion-exchangechromatography can be also used to separate peptides based on theircharge.

It will be appreciated, of course, that the peptides or antibodies,derivatives, or fragments thereof may incorporate amino acid residueswhich are modified without affecting activity. For example, the terminimay be derivatized to include blocking groups, i.e. chemicalsubstituents suitable to protect and/or stabilize the N- and C-terminifrom “undesirable degradation”, a term meant to encompass any type ofenzymatic, chemical or biochemical breakdown of the compound at itstermini which is likely to affect the function of the compound, i.e.sequential degradation of the compound at a terminal end thereof.

Blocking groups include protecting groups conventionally used in the artof peptide chemistry which will not adversely affect the in vivoactivities of the peptide. For example, suitable N-terminal blockinggroups can be introduced by alkylation or acylation of the N-terminus.Examples of suitable N-terminal blocking groups include C1-05 branchedor unbranched alkyl groups, acyl groups such as formyl and acetylgroups, as well as substituted forms thereof, such as theacetamidomethyl (Acm) group. Desamino analogs of amino acids are alsouseful N-terminal blocking groups, and can either be coupled to theN-terminus of the peptide or used in place of the N-terminal reside.Suitable C-terminal blocking groups, in which the carboxyl group of theC-terminus is either incorporated or not, include esters, ketones oramides. Ester or ketone-forming alkyl groups, particularly lower alkylgroups such as methyl, ethyl and propyl, and amide-forming amino groupssuch as primary amines (—NH2), and mono- and di-alkylamino groups suchas methylamino, ethylamino, dimethylamino, diethylamino,methylethylamino and the like are examples of C-terminal blockinggroups. Descarboxylated amino acid analogues such as agmatine are alsouseful C-terminal blocking groups and can be either coupled to thepeptide's C-terminal residue or used in place of it. Further, it will beappreciated that the free amino and carboxyl groups at the termini canbe removed altogether from the peptide to yield desamino anddescarboxylated forms thereof without affect on peptide activity.

Other modifications can also be incorporated without adversely affectingthe activity and these include, but are not limited to, substitution ofone or more of the amino acids in the natural L-isomeric form with aminoacids in the D-isomeric form. Thus, the peptide may include one or moreD-amino acid resides, or may comprise amino acids which are all in theD-form. Retro-inverso forms of peptides in accordance with the presentinvention are also contemplated, for example, inverted peptides in whichall amino acids are substituted with D-amino acid forms.

Amino Acid Substitutions

In certain embodiments, the disclosed methods and compositions mayinvolve preparing peptides with one or more substituted amino acidresidues.

In various embodiments, the structural, physical and/or therapeuticcharacteristics of peptide sequences may be optimized by replacing oneor more amino acid residues.

Other modifications can also be incorporated without adversely affectingthe activity and these include, but are not limited to, substitution ofone or more of the amino acids in the natural L-isomeric form with aminoacids in the D-isomeric form. Thus, the peptide may include one or moreD-amino acid resides, or may comprise amino acids which are all in theD-form. Retro-inverso forms of peptides in accordance with the presentinvention are also contemplated, for example, inverted peptides in whichall amino acids are substituted with D-amino acid forms.

The skilled artisan will be aware that, in general, amino acidsubstitutions in a peptide typically involve the replacement of an aminoacid with another amino acid of relatively similar properties (i.e.,conservative amino acid substitutions). The properties of the variousamino acids and effect of amino acid substitution on protein structureand function have been the subject of extensive study and knowledge inthe art. For example, one can make the following isosteric and/orconservative amino acid changes in the parent polypeptide sequence withthe expectation that the resulting polypeptides would have a similar orimproved profile of the properties described above:

Substitution of alkyl-substituted hydrophobic amino acids: includingalanine, leucine, isoleucine, valine, norleucine, S-2-aminobutyric acid,S-cyclohexylalanine or other simple alpha-amino acids substituted by analiphatic side chain from CI-10 carbons including branched, cyclic andstraight chain alkyl, alkenyl or alkynyl substitutions.

Substitution of aromatic-substituted hydrophobic amino acids: includingphenylalanine, tryptophan, tyrosine, biphenylalanine, 1-naphthylalanine,2-naphthylalanine, 2-benzothienylalanine, 3-benzothienylalanine,histidine, amino, alkylamino, dialkylamino, aza, halogenated (fluoro,chloro, bromo, or iodo) or alkoxy-substituted forms of the previouslisted aromatic amino acids, illustrative examples of which are: 2-,3-or 4-aminophenylalanine, 2-,3- or 4-chlorophenylalanine, 2-,3- or4-methylphenylalanine, 2-,3- or 4-methoxyphenylalanine, 5-amino-,5-chloro-, 5-methyl- or 5-methoxytryptophan, 2′-, 3′-, or 4′-amino-,2′-, 3′-, or 4′-chloro-, 2,3, or 4-biphenylalanine, 2′,-3′,- or4′-methyl-2, 3 or 4-biphenylalanine, and 2- or 3-pyridylalanine.

Substitution of amino acids containing basic functions: includingarginine, lysine, histidine, ornithine, 2,3-diaminopropionic acid,homoarginine, alkyl, alkenyl, or aryl-substituted (from Ci-Cio branched,linear, or cyclic) derivatives of the previous amino acids, whether thesubstituent is on the heteroatoms (such as the alpha nitrogen, or thedistal nitrogen or nitrogens, or on the alpha carbon, in the pro-Rposition for example. Compounds that serve as illustrative examplesinclude: N-epsilon-isopropyl-lysine, 3-(4-tetrahydropyridyl)-glycine,3-(4-tetrahydropyridyl)-alanine, N,N-gamma, gamma′-diethyl-homoarginine.Included also are compounds such as alpha methyl arginine, alpha methyl2,3-diaminopropionic acid, alpha methyl histidine, alpha methylornithine where alkyl group occupies the pro-R position of the alphacarbon. Also included are the amides formed from alkyl, aromatic,heteroaromatic (where the heteroaromatic group has one or morenitrogens, oxygens, or sulfur atoms singly or in combination) carboxylicacids or any of the many well-known activated derivatives such as acidchlorides, active esters, active azolides and related derivatives) andlysine, ornithine, or 2,3-diaminopropionic acid.

Substitution of acidic amino acids: including aspartic acid, glutamicacid, homoglutamic acid, tyrosine, alkyl, aryl, arylalkyl, andheteroaryl sulfonamides of 2,4-diaminopriopionic acid, ornithine orlysine and tetrazole-substituted alkyl amino acids.

Substitution of side chain amide residues: including asparagine,glutamine, and alkyl or aromatic substituted derivatives of asparagineor glutamine.

Substitution of hydroxyl containing amino acids: including serine,threonine, homoserine, 2,3-diaminopropionic acid, and alkyl or aromaticsubstituted derivatives of serine or threonine. It is also understoodthat the amino acids within each of the categories listed above can besubstituted for another of the same group.

For example, the hydropathic index of amino acids may be considered(Kyte & Doolittle, 1982, J. Mol. Biol, 157: 105-132). The relativehydropathic character of the amino acid contributes to the secondarystructure of the resultant protein, which in turn defines theinteraction of the protein with other molecules. Each amino acid hasbeen assigned a hydropathic index on the basis of its hydrophobicity andcharge characteristics (Kyte & Doolittle, 1982), these are: isoleucine(+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8);cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine(−0.4); threonine (−0.7); serine (−0.8); tryptophan (−0.9); tyrosine(−1.3); proline (−1.6); histidine (−3.2); glutamate (−3.5); glutamine(−3.5); aspartate (−3.5); asparagine (−3.5); lysine (−3.9); and arginine(−4.5). In making conservative substitutions, the use of amino acidswhose hydropathic indices are within +1−2 is preferred, within +/−1 aremore preferred, and within +/−0.5 are even more preferred.

Amino acid substitution may also take into account the hydrophilicity ofthe amino acid residue (e.g., U.S. Pat. No. 4,554,101). Hydrophilicityvalues have been assigned to amino acid residues: arginine (+3.0);lysine (+3.0); aspartate (+3.0); glutamate (+3.0); serine (+0.3);asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (−0.4);proline (−0.5+−0.1); alanine (−0.5); histidine (−0.5); cysteine (−1.0);methionine (−1.3); valine (−1.5); leucine (−1.8); isoleucine (−1.8);tyrosine (−2.3); phenylalanine (−2.5); tryptophan (−3.4). Replacement ofamino acids with others of similar hydrophilicity is preferred.

Other considerations include the size of the amino acid side chain. Forexample, it would generally not be preferred to replace an amino acidwith a compact side chain, such as glycine or serine, with an amino acidwith a bulky side chain, e.g., tryptophan or tyrosine. The effect ofvarious amino acid residues on protein secondary structure is also aconsideration. Through empirical study, the effect of different aminoacid residues on the tendency of protein domains to adopt analpha-helical, beta-sheet or reverse turn secondary structure has beendetermined and is known in the art (see, e.g., Chou & Fasman, 1974,Biochemistry, 13:222-245; 1978, Ann. Rev. Biochem., 47: 251-276; 1979,Biophys. J., 26:367-384).

Based on such considerations and extensive empirical study, tables ofconservative amino acid substitutions have been constructed and areknown in the art. For example: arginine and lysine; glutamate andaspartate; serine and threonine; glutamine and asparagine; and valine,leucine and isoleucine. Alternatively: Ala (A) leu, ile, val; Arg (R)gin, asn, lys; Asn (N) his, asp, lys, arg, gin; Asp (D) asn, glu; Cys(C) ala, ser; Gin (Q) glu, asn; Glu (E) gin, asp; Gly (G) ala; His (H)asn, gin, lys, arg; Ile (I) val, met, ala, phe, leu; Leu (L) val, met,ala, phe, ile; Lys (K) gin, asn, arg; Met (M) phe, ile, leu; Phe (F)leu, val, ile, ala, tyr; Pro (P) ala; Ser (S), thr; Thr (T) ser; Trp (W)phe, tyr; Tyr (Y) trp, phe, thr, ser; Val (V) ile, leu, met, phe, ala.

Other considerations for amino acid substitutions include whether or notthe residue is located in the interior of a protein or is solventexposed. For interior residues, conservative substitutions wouldinclude: Asp and Asn; Ser and Thr; Ser and Ala; Thr and Ala; Ala andGly; Ile and Val; Val and Leu; Leu and Ile; Leu and Met; Phe and Tyr;Tyr and Trp. (See, e.g., PROWL Rockefeller University website). Forsolvent exposed residues, conservative substitutions would include: Aspand Asn; Asp and Glu; Glu and Gin; Glu and Ala; Gly and Asn; Ala andPro; Ala and Gly; Ala and Ser; Ala and Lys; Ser and Thr; Lys and Arg;Val and Leu; Leu and Ile; Ile and Val; Phe and Tyr. Various matriceshave been constructed to assist in selection of amino acidsubstitutions, such as the PAM250 scoring matrix, Dayhoff matrix,Grantham matrix, McLachlan matrix, Doolittle matrix, Henikoff matrix,Miyata matrix, Fitch matrix, Jones matrix, Rao matrix, Levin matrix andRisler matrix (Idem.)

In determining amino acid substitutions, one may also consider theexistence of intermolecular or intramolecular bonds, such as formationof ionic bonds (salt bridges) between positively charged residues (e.g.,His, Arg, Lys) and negatively charged residues (e.g., Asp, Glu) ordisulfide bonds between nearby cysteine residues.

Methods of substituting any amino acid for any other amino acid in anencoded peptide sequence are well known and a matter of routineexperimentation for the skilled artisan, for example by the technique ofsite-directed mutagenesis or by synthesis and assembly ofoligonucleotides encoding an amino acid substitution and splicing intoan expression vector construct.

Acid addition salts of the present invention are also contemplated asfunctional equivalents. Thus, a peptide in accordance with the presentinvention treated with an inorganic acid such as hydrochloric,hydrobromic, sulfuric, nitric, phosphoric, and the like, or an organicacid such as an acetic, propionic, glycolic, pyruvic, oxalic, malic,malonic, succinic, maleic, fumaric, tataric, citric, benzoic, cinnamie,mandelic, methanesulfonic, ethanesulfonic, p-toluenesulfonic, salicyclicand the like, to provide a water soluble salt of the peptide is suitablefor use in the invention.

The present invention also provides for analogs of proteins. Analogs candiffer from naturally occurring proteins or peptides by conservativeamino acid sequence differences or by modifications which do not affectsequence, or by both.

For example, conservative amino acid changes may be made, which althoughthey alter the primary sequence of the protein or peptide, do notnormally alter its function. To that end, 10 or more conservative aminoacid changes typically have no effect on peptide function.

Modifications (which do not normally alter primary sequence) include invivo, or in vitro chemical derivatization of polypeptides, e.g.,acetylation, or carboxylation. Also included are modifications ofglycosylation, e.g., those made by modifying the glycosylation patternsof a polypeptide during its synthesis and processing or in furtherprocessing steps; e.g., by exposing the polypeptide to enzymes whichaffect glycosylation, e.g., mammalian glycosylating or deglycosylatingenzymes. Also embraced are sequences which have phosphorylated aminoacid residues, e.g., phosphotyrosine, phosphoserine, orphosphothreonine.

Also included are polypeptides or antibody fragments which have beenmodified using ordinary molecular biological techniques so as to improvetheir resistance to proteolytic degradation or to optimize solubilityproperties or to render them more suitable as a therapeutic agent.Analogs of such polypeptides include those containing residues otherthan naturally occurring L-amino acids, e.g., D-amino acids ornon-naturally occurring synthetic amino acids. The peptides of theinvention are not limited to products of any of the specific exemplaryprocesses listed herein.

Substantially pure protein obtained as described herein may be purifiedby following known procedures for protein purification, wherein animmunological, enzymatic or other assay is used to monitor purificationat each stage in the procedure. Protein purification methods are wellknown in the art, and are described, for example in Deutscher et al.(ed., 1990, Guide to Protein Purification, Harcourt Brace Jovanovich,San Diego). In another embodiment disclosed herein, peptide longevity isenhanced by the addition of adducts such as sucrose or polyethyleneglycol, production of peptide-IgG chimeras, or the peptides can becyclized via cysteine-cysteine linkages, which is a modification knownto enhance the biological activities of a variety of peptides.

In one aspect a polyethylene glycol adduct is(2-aminoethyl)-O′-(N-diglycolyl-2-aminoethyl)-hexaethyleneglycol. Inanother aspect of the invention, a polyethylene glycol adduct is in theform ofGK[(2-aminoethyl)-O′-(N-diglycolyl-2-aminoethyl)-hexaethyleneglycol]GG.The dipeptide GK increases peptide solubility. The dipeptide GG ispresent as a spacer between the solid support and peptide chain toimprove the ease of peptide synthesis.

The present disclosure also contemplates any of the peptides derivatizedwith functional groups and/or linked to other molecules to facilitatetheir delivery to specific sites of action, to potentiate theiractivity, or complexed covalently or non-covalently to otherpharmaceuticals, bioactive agents, or other molecules. Suchderivatizations must be accomplished so as to not significantlyinterfere with the properties of the peptides. Carriers andderivatizations must also be designed or chosen so as not to exert toxicor undesirable activities on animals or humans treated with theseformulations. Functional groups which may be covalently linked to thepeptides may include, but not be limited to, amines, alcohols, orethers. Functional groups to be covalently linked to the peptides toincrease their in vivo half-lives may include, but not be limited to,polyethylene glycols, small carbohydrates such as sucrose, or peptidesand proteins. The peptides may also be synthesized by recombinant DNAtechniques with expression vectors for use in biological systems, suchas bacteria, yeast, insect, or mammalian cells.

Generally, the amount of peptide administered depends upon the degree ofimmune response that is desired. Those skilled in the art may deriveappropriate dosages and schedules of administration to suit the specificcircumstances and needs of the patient. Typically, dosages of peptideare between about 0.001 mg/kg and about 100 mg/kg body weight. In someembodiments dosages are between about 0.01 mg/kg and about 60 mg/kg bodyweight. In other embodiments, dosages are between about 0.05 mg/kg andabout 5 mg/kg body weight.

In general, the schedule or timing of administration of a peptide of theinvention is according to the accepted practice for the procedure beingperformed.

When used in vivo, the peptides of the invention are preferablyadministered as a pharmaceutical composition. The invention thusprovides pharmaceutical compositions comprising a peptide, or apharmaceutically acceptable salt thereof, and a pharmaceuticallyacceptable carrier. The peptide of the invention may be present in apharmaceutical composition in an amount from 0.001 to 99.9 wt %, andmore preferably from about 0.1 to 99.0 wt %. To achieve good plasmaconcentrations, a peptide or a combination of peptides, may beadministered, for example, by intravenous injection, as a solutioncomprising 0.1 to 1.0% of the active agent.

The compositions of the present invention may comprise at least oneactive peptide, one or more acceptable carriers, and optionally otherpeptides or therapeutic agents.

For in vivo applications, the peptides of the present invention maycomprise a pharmaceutically acceptable salt. Suitable acids which arecapable of forming such salts with the compounds of the presentinvention include inorganic acids such as hydrochloric acid, hydrobromicacid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid,phosphoric acid and the like; and organic acids such as formic acid,acetic acid, propionic acid, glycolic acid, lactic acid, anthranilicacid, cinnamic acid, naphthalene sulfonic acid, sulfanilic acid and thelike.

Pharmaceutically acceptable carriers include physiologically tolerableor acceptable diluents, excipients, solvents or adjuvants. Thecompositions are preferably sterile and nonpyrogenic. Examples ofsuitable carriers include, but are not limited to, water, normal saline,dextrose, mannitol, lactose or other sugars, lecithin, albumin, sodiumglutamate, cysteine hydrochloride, ethanol, polyols (propylene glycol,polyethylene glycol, glycerol, and the like), vegetable oils (such asolive oil), injectable organic esters such as ethyl oleate, ethoxylatedisosteraryl alcohols, polyoxy ethylene sorbitol and sorbitan esters,microcrystalline cellulose, aluminum methahydroxide, bentonite, kaolin,agar-agar and tragacanth, or mixtures of these substances, and the like.

The pharmaceutical compositions may also contain minor amounts ofnontoxic auxiliary pharmaceutical substances or excipients and/oradditives, such as wetting agents, emulsifying agents, pH bufferingagents, antibacterial and antifungal agents (such as parabens,chlorobutanol, phenol, sorbic acid, and the like). Suitable additivesinclude, but are not limited to, physiologically biocompatible buffers(e.g., tromethamine hydrochloride), additions (e.g., 0.01 to 10 molepercent) of chelants (such as, for example, DTPA or DTPA-bisamide) orcalcium chelate complexes (as for example calcium DTPA orCaNaDTPA-bisamide), or, optionally, additions (e.g. 1 to 50 molepercent) of calcium or sodium salts (for example, calcium chloride,calcium ascorbate, calcium gluconate or calcium lactate). If desired,absorption enhancing or delaying agents (such as liposomes, aluminummonostearate, or gelatin) may be used. The compositions can be preparedin conventional forms, either as liquid solutions or suspensions, solidforms suitable for solution or suspension in liquid prior to injection,or as emulsions. Pharmaceutical compositions according to the presentinvention can be prepared in a manner fully within the skill of the art.

The peptides of the invention, pharmaceutically acceptable saltsthereof, or pharmaceutical compositions comprising these compounds maybe administered so that the compounds may have a physiological effect.Administration may occur enterally or parenterally; for example orally,rectally, intracisternally, intravaginally, intraperitoneally, locally(e.g., with powders, ointments or drops), or as a buccal or nasal sprayor aerosol. Parenteral administration is preferred. Particularlypreferred parenteral administration methods include intravascularadministration (e.g. intravenous bolus injection, intravenous infusion,intra-arterial bolus injection, intra-arterial infusion and catheterinstillation into the vasculature), peri- and intra-target tissueinjection (e.g. peri-tumoral and intra-tumoral injection), subcutaneousinjection or deposition including subcutaneous infusion (such as byosmotic pumps), intramuscular injection, and direct application to thetarget area, for example by a catheter or other placement device.

Where the administration of the peptide is by injection or directapplication, the injection or direct application may be in a single doseor in multiple doses. Where the administration of the compound is byinfusion, the infusion may be a single sustained dose over a prolongedperiod of time or multiple infusions.

A composition of the invention may comprise additional ingredients. Asused herein, “additional ingredients” include, but are not limited to,one or more of the following: excipients; surface active agents;dispersing agents; inert diluents; granulating and disintegratingagents; binding agents; lubricating agents; sweetening agents; flavoringagents; coloring agents; preservatives; physiologically degradablecompositions such as gelatin; aqueous vehicles and solvents; oilyvehicles and solvents; suspending agents; dispersing or wetting agents;emulsifying agents, demulcents; buffers; salts; thickening agents;fillers; emulsifying agents; antioxidants; antibiotics; antifungalagents; stabilizing agents; and pharmaceutically acceptable polymeric orhydrophobic materials. Other “additional ingredients” which may beincluded in the pharmaceutical compositions of the invention are knownin the art and described, for example in Genaro, ed., 1985, Remington'sPharmaceutical Sciences, Mack Publishing Co., Easton, Pa., which isincorporated herein by reference.

The pharmaceutical composition may be administered to an animal asfrequently as several times daily, or it may be administered lessfrequently, such as once a day, once a week, once every two weeks, oncea month, or even lees frequently, such as once every several months oreven once a year or less. The frequency of the dose will be readilyapparent to the skilled artisan and will depend upon any number offactors, such as, but not limited to, the type and severity of thecondition or disease being treated, the type and age of the animal, etc.

Typically, dosages of the compound of the invention which may beadministered to an animal, preferably a human, range in amount from 1 μgto about 100 g per kilogram of body weight of the animal. While theprecise dosage administered will vary depending upon any number offactors, including but not limited to, the type of animal and type ofdisease state being treated, the age of the animal and the route ofadministration. In one aspect, the dosage of the compound will vary fromabout 1 mg to about 10 g per kilogram of body weight of the animal. Inanother aspect, the dosage will vary from about 10 mg to about 1 g perkilogram of body weight of the animal. The compound may be administeredto an animal as frequently as several times daily, or it may beadministered less frequently, such as once a day, once a week, onceevery two weeks, once a month, or even lees frequently, such as onceevery several months or even once a year or less. The frequency of thedose will be readily apparent to the skilled artisan and will dependupon any number of factors, such as, but not limited to, the type ofcancer being diagnosed, the type and severity of the condition ordisease being treated, the type and age of the animal, etc.

Suitable preparations include injectables, either as liquid solutions orsuspensions, however, solid forms suitable for solution in, suspensionin, liquid prior to injection, may also be prepared. The preparation mayalso be emulsified, or the polypeptides encapsulated in liposomes. Theactive ingredients are often mixed with excipients which arepharmaceutically acceptable and compatible with the active ingredient.Suitable excipients are, for example, water saline, dextrose, glycerol,ethanol, or the like and combinations thereof. In addition, if desired,the vaccine preparation may also include minor amounts of auxiliarysubstances such as wetting or emulsifying agents, pH buffering agents,and/or adjuvants.

The invention also includes a kit comprising the composition of theinvention and an instructional material which describes adventitiallyadministering the composition to a cell or a tissue of a subject. Inanother embodiment, this kit comprises a (preferably sterile) solventsuitable for dissolving or suspending the composition of the inventionprior to administering the compound to the subject.

As used herein, an “instructional material” includes a publication, arecording, a diagram, or any other medium of expression which can beused to communicate the usefulness of the peptide of the invention inthe kit for effecting alleviation of the various diseases or disordersrecited herein. Optionally, or alternately, the instructional materialmay describe one or more methods of using the compositions fordiagnostic or identification purposes or of alleviation the diseases ordisorders in a cell or a tissue of a mammal. The instructional materialof the kit of the invention may, for example, be affixed to a containerwhich contains the multimeric peptide of the invention or be shippedtogether with a container which contains the peptide. Alternatively, theinstructional material may be shipped separately from the container withthe intention that the instructional material and the compound be usedcooperatively by the recipient.

In other embodiments, therapeutic agents, including, but not limited to,cytotoxic agents, anti-angiogenic agents, pro-apoptotic agents,antibiotics, hormones, hormone antagonists, chemokines, drugs, prodrugs,toxins, enzymes or other agents may be used as adjunct therapies.

Nucleic acids useful in the present invention include, by way of exampleand not limitation, oligonucleotides and polynucleotides such asantisense DNAs and/or RNAs; ribozymes; DNA for gene therapy; viralfragments including viral DNA and/or RNA; DNA and/or RNA chimeras; mRNA;plasmids; cosmids; genomic DNA; cDNA; gene fragments; various structuralforms of DNA including single-stranded DNA, double-stranded DNA,supercoiled DNA and/or triple-helical DNA; Z-DNA; and the like. Thenucleic acids may be prepared by any conventional means typically usedto prepare nucleic acids in large quantity. For example, DNAs and RNAsmay be chemically synthesized using commercially available reagents andsynthesizers by methods that are well-known in the art (see, e.g., Gait,1985, OLIGONUCLEOTIDE SYNTHESIS: A PRACTICAL APPROACH (IRL Press,Oxford, England)). RNAs may be produce in high yield via in vitrotranscription using plasmids such as SP65 (Promega Corporation, Madison,Wis.).

The invention further provides a kit comprising one or more peptides orexpression vectors of the invention, an applicator, an instructionalmaterial for the use thereof.

Other embodiments of the invention will be apparent to those skilled inthe art based on the disclosure and embodiments of the inventiondescribed herein. It is intended that the specification and examples beconsidered as exemplary only, with a true scope and spirit of theinvention being indicated by the following claims. While somerepresentative experiments have been performed in test animals, similarresults are expected in humans. The exact parameters to be used forinjections in humans can be easily determined by a person skilled in theart.

The invention is now described with reference to the following Examplesand Embodiments. Without further description, it is believed that one ofordinary skill in the art can, using the preceding description and thefollowing illustrative examples, make and utilize the present inventionand practice the claimed methods. The following working examplestherefore, are provided for the purpose of illustration only andspecifically point out the preferred embodiments of the presentinvention, and are not to be construed as limiting in any way theremainder of the disclosure. Therefore, the examples should be construedto encompass any and all variations which become evident as a result ofthe teaching provided herein.

Examples

1. IL-2 regulates inflammation in the Pancreas of mice independent ofTregs. (A) The Foxp3 mutant scurfy (Sf) mice are completely deficient inTreg cells. Yet they are resistant to inflammation in pancreas despiteinflammation in several other organs. An additional deficiency of IL-2in Sf mice (SfJ/2″/_) results in inflammation in pancreas. The IL-2deficient mice with a partial Treg deficiency (7/2/_) also develop severinflammation in pancreas. Islets are marked with ellipses andinflammation is shown with arrows. (B) The pancreas infiltrating cellsproduce mostly IFNy and little IL-4 as measured by intracellularstaining and flow cytometry on ex vivo restimulation with PMA andIonomicin. (C) Further, deletion of Th2 response genes IL-4 (SfJ/4/_) orSTATE (Sf.Stat6′/′), also leads to severe pancreatitis in Treg-deficientmice. Deficiency of IFNy (Sfffng{circumflex over ( )}′) results inprotection against such inflammation. Islets are marked with ellipsesand inflammation is shown with arrows. (See FIGS. 1A-1C).

2. IL-2 is a negative regulator of T follicular helper (TfH)differentiation. The TfH cells produce IL-21, promote plasma celldifferentiation and production of high affinity autoantibodies. (A)Deficiency of IL-2 induces the expression of genes related to TfHprogram on CD4+ T cells. (B) This is accompanied with infiltration ofplasma cells (arrow) in the pancreas as measured by flow cytometry onthe cells isolated form the pancreas. (See FIGS. 2A and 2B).

3. The natural Tregs (nTr) express IL-33 receptor (IL1RL1). (A). IL1RL1expression by real-time PCR was measured by real-time PCR on FACS sortednTr cells. FACS sorted naive cells (Tn) were differentiated in vitrointo induced Tregs (iTr), effector cells (Teff), Th1 or Th2 cells andanalyzed for IL1RL1 expression. FACS sorted naive T-cells (Tn) were usedas a control. (B) Expression of ILIRLI as gated on Tregs and non-Tregswas analyzed by flow cytometry. (See FIGS. 3A and 3B).

4. Treatment with IL233 increases natural Tregs (Foxp6⁺ Helios⁺) inmice. Non-obese diabetic (NOD) mice were injected with five daily dosesof 1.0 μg molar equivalent of a combination of IL-2 and IL-33 or IL233and CD4 Foxp3 Helios+ cells were evaluated in the peripheral blood byflow cytometry. The control mice were injected with saline. Mice weretypically about 20 grams. (See FIG. 4).

5. Treatment of mice with IL233 protects non-obese diabetic (NOD) micefrom type-1 diabetes like disease. (A) Female 20 week old NOD mice weretreated for five days with daily i.p. injections of a mixture of 1.0 μgeach of IL-2 and IL-33 (orange) or IL233 (green; molar equivalent to 1.0μg IL-2) before the onset of hyperglycemia. The treatment with thecytokines protected the mice for long-term against onset of type-1diabetes like disease as compared to untreated controls (blue). (B) NODfemales that were early diabetic were treated for 5 days with daily i.p.injections of 1 μg IL-2 molar equivalent of recombinant IL-2 (red) orIL233 (green). Treatment with IL233, but not IL-2 alone, protected themice from ongoing hyperglycemia as shown by the delay in the diseasekinetics with 2/5 mice showing complete protection. Blue arrow indicatesstart of the treatment. (C) In another experiment mice with earlydiabetes (blood glucose of 150+10 mg/dL) were treated with one time with5-daily injections of 0.3 μg/mouse molar equivalent of IL-2 alone, IL-33alone, a combination of IL-2 and IL-33, or IL233. The control mice wereinjected with saline only. The numbers in parenthesis on the rightrepresent the number of mice in each group. (See FIGS. 5A-5C).

6. Treatment with IL233 expands Treg cells especially in the pancreaticLymph nodes.

Non-obese diabetic mice with early diabetes (blood glucose 150+10 mg/dL)were injected once with a 5-daily dose of 1.0 μg equivalent IL-2 orIL-33 or combination of IL-2 and IL-33 or IL233 hybrid cytokine. Thecontrol mice were injected with saline. Six weeks post-treatment, themice that recovered from hyperglycemia or the mice that were severelydiabetic (blood glucose of 600 mg/dL for two consecutive days) wereeuthanized and the spleen and pancreatic lymph nodes were analyzed forthe CD4+Foxp3+Treg cells. (See FIG. 6).

7. Treatment with IL233 or mixture of IL-2 and IL-33 protects C57BL/6mice from renal ischemia reperfusion injury (IRI). C57BL/6 mice weretreated with 5 daily doses of the indicated amounts of a combination ofmurine IL-2 (m2) and IL-33 (m33) or with murine IL233 fusion cytokine(m233). On day 7, the renal pedicle was clamped for 26 minutes followedby 18 hours reperfusion. High plasma creatinine levels indicate loss ofkidney function. Unexpectedly, the IL233 fusion was 3-fold moreeffective than the mixture of IL-2 and IL-33 in protecting mice againstloss of renal function and inflammation as measure by Plasma creatininelevels (A) or Histological score. (B). The protection was accompaniedwith reduction of the activated CD4+ T cells (C), CD8+ T-cells (notshown) and activated B-cells (D) in the spleen. TNFa producing CD4 (E)and CD8 (F) T-cells and IFN-γ producing cells (not shown) were alsoreduced by the cytokine treatment as measured by flow cytometry of thecells isolated from the spleen. (See FIGS. 7A-7F).

8. Treatment with IL233 protects lupus prone NZM2328 mice from lupusglomerulonephritis (GN). Three-month-old NZM2328 female mice (5 mice pergroup) were injected with recombinant IL-2 or IL233 fusion protein({circumflex over ( )}gIL-2 molar equivalent of each) daily for 5 days(green arrows). The control mice received saline only. On day 12 all themice with injected with Ad-IFNa (adenovirus expressing IFNa) toaccelerate the lupus nephritis (red double-headed arrow). The mice weremonitored periodically for kidney function (proteinuria with dip stick)and mortality. (A) The peripheral blood of mice was analyzed on dl andd9 of the recombinant cytokine treatment. Treatment with both IL-2 andIL233 resulted in an increase in the Treg levels, as measured by flowcytometry. Treatment with IL233, but not IL-2 resulted in a decrease inthe production of TNF-a (a pro-inflammatory cytokine known to contributeto lupus GN) as measured by intracellular staining of lymphocytes forTNF-a after a 5-hour ex-vivo stimulation with Phorbol myristate acetateand Ionomycin. Individual mice are shown. (B) Proteinuria was measuredby dipstick and mice with a “+++” score on the dipstick were consideredto have severe proteinuria (top). The data is presented as “percentproteinuria free”. The control mice (Blue) developed severe proteinuriarapidly, while treatment with IL-2 (red) protected partially. The micetreated with IL233 (green) were completely protected against severeproteinuria at the termination of the experiment. IL233 treatmentoffered complete protection against mortality, which IL-2 treatmentprotected partially (bottom). (See FIGS. 8A and 8B).

9. Treatment with IL233 as well as the IL-2 and IL-33 combination ismore effective than either cytokine alone to protect lupus prone NZM2328mice from GN. In a similar experiment, 3-month old NZM2328 mice wereinjected for 5 consecutive days (green arrow) with 1 μg molar equivalentdaily of IL-2, IL-33, a mixture of IL-2 and IL-33, or IL233. Three dayslater mice were injected with Adenovirus expressing IFNa to acceleratelupus GN. A) Combined treatment with IL-2 and IL-33, especially as aIL233 cytokine protected NZM2328 mice from severe proteinuria (top) andmortality (bottom) in IFNa-induced accelerated GN (green arrows-cytokinetreatment; red arrows IFNa; n=5). B) Representative H & E stained kidneysections show enlarged glomeruli (highlighted by black-dotted circle;higher magnification in the inset), mesangial expansion,glomerulosclerosis (inset) and leukocytic infiltration in the controlmice, but not in the IL233-treatment group (quantified in C). D) IL233treatment, although inhibited glomerular hypertrophy, did notsignificantly alter Complement C3 and total IgG immune complexdeposition. E) IL233 treatment skewed the circulating anti-dsDNAantibodies from IgG2a to IgG2b, p<0.01; n=3. IL233 treatment increasedFoxp3+ Tregs as measured in the lymph node (LN) of mice (F) leading tolower ratios of Tregs to IFN+ (G) & TNF+ (H) CD4 T-cells when analyzed12-wks post initial treatment or when the control of IL-2 or IL-33treated mice were moribund. ♦=individual mice; bar=mean. (See FIGS.9A-9H).

10. Treatment with IL233 inhibits progression of obesity, type-2diabetes (T2D) and diabetic nephropathy in mice genetically predisposedfor obesity. Five to six weeks old BTBR.ob/ob (Ob-obese mice, due tomutation in Leptin gene) or BTBR.ob/+ (Het, non-obese mice) were treatedonce with 5-daily doses of 50 μg/kg of IL233 or saline (green arrows).The mice were monitored for CD4+Foxp3+ Tregs (A), body weight (B), bloodglucose (C), proteinuria (D). The glucose tolerance of the mice treatedwith IL233 also improved to near non-obese levels (E). As shown belowIL233 treatment increased the Treg levels, inhibited weight gain,hyperglycemia, and proteinuria, and restored glucose tolerance. (SeeFIGS. 10A-10E).

11. Preparation of Human and Murine IL233 Fusion Proteins

The nucleotide sequences of the nucleic acids encoding the fusionproteins and the amino acid sequences of the recombinant IL233 fusionproteins are depicted in FIGS. 11A-11D. The Blue color denotes the IL-2coding sequence, the red color is the IL-33 encoding sequence, the blackresidues are the linker segments added to provide flexibility to thefusion protein, and the yellow highlighted part codes for the TEVprotease cleave site. The major restriction sites are underlined. (SeeFIGS. 11A-11D). The sequences are also provided in the Sequence Listingand in the Summary of the Invention.

We synthesized nucleic acids comprising nucleic acid sequences encodinghuman and murine IL-2 (human amino acid residues 21-153; murine aminoacid residues 21-169) and IL-33 (human amino acid residues 112-270;murine amino acid residues 109-266) that were optimized for E. colicodon bias. The cytokines were produced in E. coli and purified to nearhomogeneity.

Recombinant Human IL233 Hybrid Cytokine: (See FIGS. 11A-11D, 12A-12D,and 13) Novel hybrid cytokines were generated consisting of mature IL-2(residues 21-153) and mature IL-33 (residues 112-270) proteins separatedby a short linker (GGGGSGGGGSGGGGS) sequence (SEQ ID NO:5). The fusionprotein is named IL233 (hIL233) and was produced in E. coli. The codingsequence for the IL233 fusion protein has been synthesized usingoptimized codon sequences for high-level expression. The sequence of thesynthetic gene for the IL233 fusion protein is given in FIGS. 11A-11D.The gene was synthesized using overlapping oligonucleotides andamplified using Polymerase Chain Reaction (PCR) with Deep Vent™ DNApolymerase (New England Biolabs). The coding sequence was cloned as anin-frame fusion with NusA coding sequence in the pET44a expressionvector (Novagen Inc.), under the control of T7 promoter. This strategyresults in a fusion protein consisting of (His)6-NusA-TEVlinker-rhIL2(21-153)-linker-rhIL33 (112-270). For generating theexpression vector, the PCR product was digested with EcoRI and Xholrestriction enzymes (New England Biolabs). The pET44a vector wasdigested with EcoRI (New England Biolabs) and Xhol restriction enzymes.Both DNA fragments were purified on agarose gel and ligated to eachother using T4 DNA ligase (Bioline Inc.) and used to transform E. coliDH5a competent cells. The positive clones were selected on Luria Bertani(LB) agar plates containing 100 g/ml Ampicillin.

Recombinant Murine IL233 Hybrid (mIL233) Cytokine:

To establish the proof of concept we produced the murine version of therecombinant cytokines. The plasmid vector and techniques were the sameas for human IL233, the only difference being the use of mouse IL-2 andIL-33 genes. The gene for mouse IL-2 coding sequence was amplified byreverse transcriptase PCR using C57BL/6 (B6) spleen mRNA as a templateand the mouse IL-33 coding sequence was synthesized. The coding sequencefor mouse IL-2 (mIL-2; residues 21-169) and IL-33 (mIL-33; residues109-266) were linked with the coding sequence for a flexible linker asfor human IL233, thus generating (His)6-NusA-TEVlinker-rmIL2(21-169)-linker-rmlL-33 (109-266). The DNA and proteinsequences for the murine version of IL233 (mIL233) are given below inFIGS. 11C and 11D, respectively.

The expression vector for mouse or human IL233 was used to transform E.coli ROSETTA-GAMI™ brand competent cells (Novagen Inc.). The expressionof the protein was induced with 0.1 mM IPTG and the protein waspartially purified using immobilized metal-affinity chromatography onNI-NTA-AGAROSE™ brand resin (Qiagen Inc.) using the manufacturer'sprotocol. The (His)6-NusA-linker was removed by digestion with TEVprotease (Sigma Aldrich Inc.) and purified using ion exchange andsize-exclusion chromatography. The final product was dialyzed againstPhosphate buffered saline (PBS), quantified using Coomassie blue reagentand stored in aliquots at −80° C. The sequence of the final fusionprotein is given in FIGS. 11A-11D.

The potential mode of action of the fusion protein can be envisioned inFIGS. 12A-12D. The IL233 fusion protein (FIG. 12A) can induceproliferation and activation cells bearing the receptors for IL-2 andIL-33 in an autologous (FIG. 12B) or fraternal manner (FIG. 12C). Thefusion protein can also induce recruitment of Treg, Th2 or ILC cells tothe sites of inflammation to either suppress the pro-inflammatory Th1,Th17 or NK cells (Fig-1 ID left) or induce tolerance by imparting a Th2skewing or altered maturation phenotype on the antigen presentingDC/macrophages (FIG. 11D, right).

We demonstrated that a treatment of mice with a combination of low-doseIL-2 and IL-33 or with the mIL233 hybrid cytokine was effective insuppressing the onset of hyperglycemia in a mouse model of type-1diabetes (T1D). We also showed that treatment of mice with thecombination of low-dose IL-2 and IL-33 as well as IL233 also offeredprotection against acute kidney injury in a mouse model of ischemiareperfusion. The IL-2 and IL-33 combination therapy and more effectivelyas the IL233 cytokine were, protective in a mouse model of lupusglomerulonephritis as compared to either cytokine alone. It is alsodemonstrated that the IL233 cytokine protected against obesity, type-2diabetes (T2D), and obesity-linked diabetic nephropathy using a mousemodel.

12. Different modes of action of the IL233 fusion protein. To provide anexample of the modes and potential mechanisms of action of the fusionprotein, a schematic representation of the IL233 fusion protein and itsreceptors and action is provided in FIGS. 12A-12D. IL233 may bind to tworeceptors on the same cell (B) or on adjacent cells bearing thereceptors for IL-2 and IL-33 (C). In a multimeric complex of cells,IL233 binding to Tregs, Th2, or ILC2 on one end and to antigenpresenting cell (DC or macrophages) on the other hand may inducetolerance resulting in suppression of Th1, TH17, or TfH activation. (SeeFIGS. 12A-12D).

13. Proposed model of using combinations of IL-2 and IL-33 or a fusionprotein of IL-2 and IL-33. Without wishing to be bound by any particulartheory, it was hypothesized herein that, owing to the constitutiveexpression of the receptors for IL-2 and IL-33 on the natural Tregs, Th2cells and Innate Lymphoid cells type 2 (ILC2), combining the activitiesof IL-2 and IL-33 could invoke multiple mechanisms for suppression ofautoimmune and inflammatory diseases. FIG. 13 provides a schematicrepresentation of the model and actions, which include: (a) activationand recruitment of Tregs/ILC2 for peripheral tolerance; (b) skewing ofthe immune response towards Th2 for suppression of the pro-inflammatoryTh1 and Th17 cells; (c) inhibiting the T-follicular helper (TfH) cells,which induce high-affinity autoantibodies; and (d) induce tolerogenicalternately activated macrophages. Owing to the high-level constitutiveexpression of the receptors for both IL-2 and IL-33, a treatment withcombination of IL-2 and IL-33 will increase the targeting of nTregs, Th2and ILC2. (See FIG. 13).

IL-33 and Treg cells: We have identified high-level expression of theIL-33 receptor ILIRLI on Treg cells (data not shown). Our data showlittle or no ILIRLI expression of naive T-cells (Tn) or when the Tn arestimulated in the absence of antigen-presenting cells (APC) and underthe conditions to induce Th1, Th2 or iTreg cells. However, ILIRLI wasconstitutively expressed at high levels on the nTreg cells and the levelwas further increased upon stimulation in the presence of IL-2 (data notshown).

CONCLUSIONS

Without wishing to be bound by any particular theory, it is hypothesizedthat IL-2 in conjunction with IL-33 is important for the maintenance andfunction of Treg cells, where IL-33 provides the innate signal, whileIL-2 provides the adaptive signal for the adequate maintenance of Treghomeostasis and function. Indeed, studies from other groups haverecently demonstrated that IL-33 can boost the Treg-mediated allograftsurvival in rodent models and also protection against experimentalcolitis (10).

The application discloses a therapy utilizing a combination ofcytokines, that simultaneously promote T-regulatory (Treg) cells andT-helper 2 (Th2 that produce IL-4, IL-5, and IL-13) and that suppressautoimmunity and inflammation by inhibiting the pro-inflammatory Th1 andTh17 cells which produce Interferon (IFN)-gamma and IL-17. The Treg,Th2, and recently discovered innate lymphoid cells (ILC2) highly expressthe receptors for IL-2 and IL-33. It is proposed herein that acombination therapy with IL-2 and IL-33 will simultaneously promote Tregand Th2 responses to offer long-term protection against autoimmunity andinflammation by suppressing the Th1 and Th17 responses as well asinhibiting activation of several other proinflammatory immune cells.

Potential Uses of the Combination Therapy and the IL233 Hybrid Cytokine:

Without wishing to bound by any particular theory, it is hypothesizedherein that that the synergy of IL-2 and IL-33 will induce multipleprotective mechanisms, which may include, but not limited to expandingand activating Tregs, Th2 and ILC2 cells, skewing of immune response andinduction of alternately activated macrophages to protect fromautoimmune and inflammatory diseases by suppressing Th1, Th17 and TfHcell responses (FIGS. 3A and 3B). Some uses of the invention are listedbelow:

1. The IL233 fusion protein can be used for treatment of inflammatoryconditions via boosting the homeostasis and activity of Treg cells,which offer the major mechanism of peripheral immune tolerance.

2. The IL233 fusion protein can be used to potentiate the Th2 responseby increasing the production of the related cytokines (IL-4, IL-5, andIL-13) by the differentiated Th2 cells, the innate lymphoid cells,Nuocytes, natural helper cells or fat associated lymphoid cluster(FALC).

3. The invention can be used for skewing of the immune response byinhibiting the pro-inflammatory Th1 response and boosting theanti-inflammatory Th2 immune response.

4. The invention can be used for altering the phenotype of innate immunecells, such as dendritic cells (DC) and macrophages to an alteredmaturation phenotype, such that these innate immune cells can skew thedifferentiation of immune response towards a Th2 response, whilesuppressing the Th1 differentiation. The IL233 treated DC andmacrophages can also be employed for boosting the homeostasis of Tregcells.

5. The function described above can either individually or incooperation be used for suppression of initiation or cure of, forexample, autoimmune disorders including Type-1 diabetes, Type-2diabetes, multiple sclerosis, atherosclerosis, systemic lupuserythematosus (lupus), autoimmune pancreatitis, IgG4-related systemicdisease spectrum diseases (IgG4-RSD), Sjogren's syndrome, inflammatorybowel disease (Crohn's disease, and ulcerative colitis), autoimmunethyroiditis, autoimmune encephalomyelitis, Alzheimer's disease &dementia, ankylosing spondylitis, chronic obstructive pulmonary disease,myasthenia gravis, obesity, osteoporosis, periodontal disease, psoriasisand uveitis.

6. The IL233 fusion protein can be employed for boosting immunetolerance during transplantation for enhancing graft survival viaboosting the homeostasis and recruitment of Treg and Th2 cells.

7. The invention can be employed for treatment of inflammatoryconditions arising due to ischemia reperfusion injury of various organsincluding, kidneys, lung and heart.

8. The IL233 treatment can be employed for protection in cardio-vasculardiseases by not only suppressing inflammation, but also by improvingcardio-vascular function. Treatment with IL-33 has been demonstrative tohave cardio-protective properties in a model of pressure overload andimproved survival following transverse aortic constriction in wild-typebut not IL-33 receptor mice. IL-33 can also reduce cardiomyocyteapoptosis, decrease infarct and fibrosis, and improve ventricularfunction in vivo via suppression of caspase-3 activity and increasedexpression of the ‘inhibitor of apoptosis’ family of proteins.

9. The IL233 fusion protein can be used to not only suppressinflammation in the central nervous system, but will also be useful inpromoting the homeostasis of microglia cells. This activity can beemployed is treatment of several neurological disorders linked withmicroglia-cell related defects such as Rhett syndrome.

10. The IL233 fusion protein can be employed to reduce inflammation inthe adipose tissue by altering the phenotype of the adipose tissueresident macrophages to an altered maturation phenotype, which willsuppress the inflammation associated with obesity and Type-2 diabetes.The fusion protein will also be useful for suppression of adipogenesis,because IL-33 has been shown to suppress the expression of several genesrelated to adipogenesis and improve fasting glucose levels as well asresistance to insulin and glucose in mouse model of type-2 diabetes.

11. The IL233 fusion protein will also suppress the inflammatorycomorbidities associated with diabetes, such as diabetic nephropathy,retinopathy, and neuropathy.

The novelty of this invention, based on the unexpected identification ofthe common high-level expression of the receptors for IL-2 and IL-33 onthe Treg cells, Th2 cells and the ILC, is the unexpected finding thatthe combination of IL-2 and IL-33 activities can be employed to boostTreg and Th2 responses to inhibit and resolve autoimmune andinflammatory diseases via suppression of the pro-inflammatory Th1, Th17and TfH responses. Additionally, the present invention now provides thedesign of a fusion protein that has the activities of both thesecytokines, which are important for immunological tolerance, in onemolecule, to improve the specificity and avidity of both the activitiesfor use as an anti-inflammatory agent. Further disclosed herein is thewide repertoire of the mode of action of the fusion protein, wherein itwas found to be successful in exploiting multiple tolerogenicmechanisms, which include, but are not limited to, regulation of Tregs,Th2 cells, ILC, Dendritic cells, macrophages, neuronal cells (microgliaand astrocytes) and adipocytes.

A common problem in treatment with soluble factors is thenon-specificity of the therapeutic approach. Current technologies forthe treatment of autoimmune and inflammatory conditions (including organtransplantation) employ strategies, which include either completeimmune-suppression or blocking of one aspect of the immune response,which either lead to infections or are ineffective respectively. Here,we propose to enhance the body's in built protective mechanisms, whichwill work on several aspects of the innate and adaptive simultaneouslyfor effective expression of immunological tolerance.

Use of IL-2 to expand regulatory T-cells either alone or as a complexwith anti-IL-2 monoclonal antibody has been used in animal models.However, since the anti-IL-2 antibody is a large foreign molecule, itwill induce immune response against itself and will soon be renderedineffective. Furthermore, such techniques only target one cell type. Thepresent invention targets multiple cell-types of immune system andemploys multiple mechanisms of immunological tolerance.

Both IL-2 and IL-33 are pleiotropic cytokines and work on multiplecell-types. Although, the highest expression of IL-2 receptor is foundon the Treg cells, IL-2 receptor is present on multiple cell types. Thehigh-affinity IL-2 receptor CD25 is also expressed on activated CD4+ andCD8+ T-cells as well as B-cells. Nevertheless, the Treg cells have thehighest constitutive expression of CD25 and consumption of IL-2 via itshigh-affinity receptor comprises one of the major mechanisms ofsuppression employed by the Tregs. The invention therefore providescompositions and methods to increase the specificity of the IL-2 andIL-33 activities as a covalently linked fusion protein, whichselectively targets the cells that are enriched for the receptors forboth. Such cells include Treg cells, Th2 cells and ILC/Nuocytes, whichhave important functions in regulation of inflammation. The inventionwill also induce the recruitment of Treg, Th2, and ILC to other celltypes, which express only one of the two receptors for IL-2 and IL-33(see FIGS. 11A-11D, 12A-12D, and 13).

The disclosures of each and every patent, patent application, andpublication cited herein are hereby incorporated by reference herein intheir entirety.

Headings are included herein for reference and to aid in locatingcertain sections. These headings are not intended to limit the scope ofthe concepts described therein under, and these concepts may haveapplicability in other sections throughout the entire specification.

While this invention has been disclosed with reference to specificembodiments, it is apparent that other embodiments and variations ofthis invention may be devised by others skilled in the art withoutdeparting from the true spirit and scope of the invention.

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1. An Interleukin-233 (IL233) fusion protein comprising a biologicallyactive domain of Interleukin-2 (IL-2) or a biologically active fragmentthereof, wherein said domain binds with the IL-2 receptor, and abiologically active domain of Interleukin-33 (IL-33) or a biologicallyactive fragment thereof, wherein said IL-33 domain binds with the IL-33receptor, further wherein said IL-2 domain is linked to said IL-33domain with a linker sequence of SEQ ID NO:5, and the fusion proteincomprises the amino acid sequences of SEQ ID NO:2 or SEQ ID NO:11. 2-3.(canceled)
 4. The fusion protein of claim 1, wherein said IL-2 comprisesthe sequence of SEQ ID NO:6 or SEQ ID NO:8 and said IL-33 comprises thesequence of SEQ ID NO:7 or SEQ ID NO:9, or biologically active fragmentsthereof.
 5. The fusion protein of claim 4, wherein said fragment of IL-2comprises the sequence of SEQ ID NO:3, and said fragment of IL-33comprises the sequence of SEQ ID NO:4, or biologically active fragmentsthereof.
 6. The fusion protein of claim 5, wherein said IL-2 comprises asequence at least 95% identical to SEQ ID NO:4.
 7. The fusion protein ofclaim 1, wherein said protein is a synthetic protein.
 8. An isolatednucleic acid comprising a nucleic acid sequence encoding a fusionprotein of claim
 1. 9. An isolated nucleic acid comprising a nucleicacid sequence encoding a fusion protein of claim
 6. 10. (canceled)
 11. Avector comprising the isolated nucleic acid of claim 9 and optionally apromoter.
 12. (canceled)
 13. A recombinant host cell comprising theisolated nucleic acid of claim
 9. 14. A recombinant host cell comprisingthe vector of claim
 11. 15. A fusion protein encoded by the isolatednucleic acid of claim
 9. 16-34. (canceled)
 35. A method of treatinginflammation associated with autoimmune diseases or disorders, saidmethod comprising administering to a subject in need thereof apharmaceutical composition comprising an effective amount of anInterleukin-233 (IL233) fusion protein of claim
 1. 36. The method ofclaim 35, wherein said pharmaceutical composition is administered atleast twice. 37-38. (canceled)
 39. The method of claim 35, wherein saidmethod stimulates proliferation of T-regulatory (Treg), Thelper2 (Th2),and innate lymphoid cells (ILC) cells and activates Treg, Th2 and ILCcells.
 40. The method of claim 39, wherein said IL233 fusion protein ora combination of Interleukin-2 (IL-2) and Interleukin-33 (IL-33)proteins are administered at a dosage ranging from about 1 μg/kg bodyweight to about 1000 μg/kg body weight. 41-44. (canceled)
 45. A methodof preventing or treating an autoimmune disease or disorder, said methodcomprising administering to a subject in need thereof a pharmaceuticalcomposition comprising an effective amount of an Interleukin-233 (IL233)fusion protein of claim
 1. 46-47. (canceled)
 48. The method of claim 45,wherein said pharmaceutical composition is administered at least twice.49-50. (canceled)
 51. The method of claim 45, wherein said methodstimulates proliferation of T-regulatory (Treg), Thelper2 (Th2), andinnate lymphoid cells (ILC) cells and activates Treg, Th2 and ILC cells.52. The method of claim 51, wherein said IL233 fusion protein or acombination of IL-2 and IL-33 proteins are administered at a dosageranging from about 1 μg/kg body weight to about 1000 μg/kg body weight.53. (canceled)
 54. A pharmaceutical composition comprising a fusionpeptide of claim 1, a pharmaceutically-acceptable carrier, andoptionally an additional therapeutic agent.
 55. A kit comprising apharmaceutical composition of claim 54, an applicator, and aninstructional material for the use thereof.